Multiaptamer target detection

ABSTRACT

Described herein are compositions comprising a first aptamer, second aptamer and a target that are capable of forming a ternary complex, and wherein the first aptamer and the second aptamer comprise C-5 pyrimidine modification schemes that are different, and methods of making and using such compositions.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/023,182, filed Mar. 18, 2016, which is a 35 U.S.C. § 371 nationalphase application of PCT/US2014/057143 (WO2015/048084), filed on Sep.24, 2014, which application claims priority under 35 U.S.C. § 119(e) toU.S. provisional application Ser. No. 61/881,629, filed on Sep. 24,2013. Each of these references is incorporated herein by reference inits entirety.

FIELD

The present disclosure relates generally to the field of nucleic acidligands, and more specifically, to aptamer pair based target detection;compositions comprising aptamer pairs and a target; and methods ofmaking and using the same.

Incorporated by reference herein in its entirety is the Sequence Listingentitled “Sequence_Listing_Amended_ST25.txt”, created Jan. 11, 2017,size of 3 kilobytes.

BACKGROUND

Protein diagnostics have a wide array of clinical application and areuseful in determining proteomic signatures or disease-specificbiomarkers. These diagnostics typically require pairs ofanalyte-specific reagents for capture and detection of the desiredtarget (e.g., protein). Antibodies have been widely used as diagnosticreagents; however they can be difficult to procure in adequate qualityand quantity, and allow only limited multiplexing when testing multipletargets. Further, they are limited in arrays for multiplexed orhigh-content proteomic applications due to their inherentcross-reactivity and non-universal assay conditions.

In contrast to antibodies, nucleic acid-based ligands have severaladvantages over antibodies including low molecular weight, thermal anddesiccation stability, reversible renaturation, ease of manufacturing,and lower cost. However, only few examples of analytes bound by twodifferent aptamers exist to date. As one example, separate DNA aptamersto the fibrinogen-recognition and heparin-binding exosites of thrombinhave been described, and both of these aptamers, TBA1 (15-mer) and TBA2(29-mer), consist of G-quartet motifs that bind to discreteelectropositive surfaces on thrombin. Sandwich assays with TBA1 and TBA2have been developed for potential thrombin monitoring, including aptamermicroarrays and fluorescence sensing platforms. Another example isintegrin α_(V)β3, for which RNA aptamers to α_(V) or β₃ subunits havebeen generated via successive selections with α_(V)β₃ or α_(IIb)β3.Aptamer pairs to TATA binding protein (TBP), prion protein (PrP), andVEGF-165 have also been reported. The limited number of aptamer pairsfor detecting protein targets is likely the result of the propensity ofaptamers to bind to predominantly cationic epitopes which drives thebest ligands to common surfaces. Thus, special selection methods havegenerally been required to force the selection toward non-overlappingepitopes.

Therefore, there continues to be a need for alternative composition andmethods for improved, cost-effective and efficient ways to detect targetproteins. The present disclosure meets such needs by providing novelcombinations of slow off-rate aptamer (SOMAmer) reagent pairs forprotein detection that comprise deoxyuridine residues modified at their5-position, which both expands the range of protein targets and improvesthe binding properties compared to conventional aptamers.

SUMMARY

The present disclosure describes a composition comprising a firstaptamer, second aptamer and a target, wherein the first aptamercomprises a first C-5 pyrimidine modification scheme, the second aptamercomprises a second C-5 pyrimidine modification scheme, and wherein thefirst C-5 pyrimidine modification scheme and the second C-5 pyrimidinemodification scheme are different; and wherein the first aptamer, secondaptamer and the target are capable of forming a ternary complex.

In another aspect of the disclosure, the first aptamer has bindingaffinity for the target and not the second aptamer.

In another aspect, the second aptamer has binding affinity for thetarget and not the first aptamer.

In another aspect, the second aptamer has binding affinity for a complexformed by the association of the first aptamer with the target.

In another aspect, the first aptamer binding region of the target andthe second aptamer binding region of the target are different regions.In a related aspect, the first aptamer and the second aptamer havenon-competing binding sites on the target.

In another aspect, the first aptamer and the second aptamer,independently, comprise RNA, DNA or a combination thereof.

In another aspect, the C-5 modified pyrimidine is selected from thegroup consisting of 5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU),5-(N-benzylcarboxyamide)-2′-O-methyluridine,5-(N-benzylcarboxyamide)-2′-fluorouridine,5-(N-phenethylcarboxyamide)-2′-deoxyuridine (PEdU),5-[N-(phenyl-3-propyl)carboxamide]-2′-deoxyuridine (PPdU),5-[N-(2-thiophene-methyl)carboxamide]-2′-deoxyuridine (ThdU) (alsoreferred to as 5-(N-thiophenylmethylcarboxyamide)-2′-deoxyuridine),5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),5-(N-isobutylcarboxyamide)-2′-O-methyluridine,5-(N-isobutylcarboxyamide)-2′-fluorouridine,5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-(N-tryptaminocarboxyamide)-2′-O-methyluridine,5-(N-tryptaminocarboxyamide)-2′-fluorouridine,5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2′-deoxyuridinechloride, 5-[N-(1-naphthylmethyl)carboxyamide]-2′-deoxyuridine (NapdU),5-[N-(2-naphthylmethyl)carboxyamide]-2′-deoxyuridine (2-NapdU),5-[N-(1-naphthylethyl)carboxyamide]-2′-deoxyuridine (NEdU),5-[N-(2-naphthylethyl)carboxyamide]-2′-deoxyuridine 2NEdU),5-[N-(4-fluorobenzyl)carboxyamide]-2′-deoxyuridine FBndU),5-[N-(4-hydroxyphenyl-2-ethyl)carboxamide]-2′-deoxyuridine (TyrdU),5-(N-naphthylmethylcarboxyamide)-2′-O-methyluridine,5-(N-naphthylmethylcarboxyamide)-2′-fluorouridine,5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine,5-[N-(3-benzo[b]thiophene-2-ethyl)carboxamide]-2′-deoxyuridine (BTdU),5-[N-(3-benzo[a]furan-2-ethyl)carboxamide]-2′-deoxyuridine (BFdU),5-[N-(3,4-methylenedioxybenzyl)carboxamide]-2′-deoxyuridine (MBndU),5-[N—((R)-2-tetrahydrofurylmethyl)carboxamide]-2′-deoxyuridine (RTHdU),5-[N—((S)-2-tetrahydrofurylmethyl)carboxamide]-2′-deoxyuridine (STHFdU),5-(N-2-imidazolylethylcarboxamide)-2′-deoxyuridine (ImiddU),5-[N-(1-morpholino-2-ethyl)carboxamide]-2′-deoxyuridine (MOEdU), and acombination thereof.

In another related aspect, the first C-5 pyrimidine modification schemecomprises a 5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU).

In another aspect, each uracil or thymine of the first aptamer is a C-5modified pyrimidine selected from the group consisting of5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU),5-(N-benzylcarboxyamide)-2′-O-methyluridine,5-(N-benzylcarboxyamide)-2′-fluorouridine,5-(N-phenethylcarboxyamide)-2′-deoxyuridine (PEdU),5-(N-thiophenylmethylcarboxyamide)-2′-deoxyuridine (ThdU),5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),5-(N-isobutylcarboxyamide)-2′-O-methyluridine,5-(N-isobutylcarboxyamide)-2′-fluorouridine,5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-(N-tryptaminocarboxyamide)-2′-O-methyluridine,5-(N-tryptaminocarboxyamide)-2′-fluorouridine,5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2′-deoxyuridinechloride, 5-[N-(1-naphthylmethyl)carboxyamide]-2′-deoxyuridine (NapdU),5-[N-(2-naphthylmethyl)carboxyamide]-2′-deoxyuridine (2-NapdU),5-(N-naphthylmethylcarboxyamide)-2′-O-methyluridine,5-(N-naphthylmethylcarboxyamide)-2′-fluorouridine and a5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine. In arelated aspect, each uracil or thymine of the first aptamer is a5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU).

In another aspect, the second C-5 pyrimidine modification schemecomprises a C-5 modified pyrimidine selected from the group consistingof 5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU),5-(N-benzylcarboxyamide)-2′-O-methyluridine,5-(N-benzylcarboxyamide)-2′-fluorouridine,5-(N-phenethylcarboxyamide)-2′-deoxyuridine (PEdU),5-(N-thiophenylmethylcarboxyamide)-2′-deoxyuridine (ThdU),5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),5-(N-isobutylcarboxyamide)-2′-O-methyluridine,5-(N-isobutylcarboxyamide)-2′-fluorouridine,5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-(N-tryptaminocarboxyamide)-2′-O-methyluridine,5-(N-tryptaminocarboxyamide)-2′-fluorouridine,5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2′-deoxyuridinechloride, 5-(N-[1-naphthylmethyl]carboxyamide)-2′-deoxyuridine (NapdU),5-(N-[2-naphthylmethyl]carboxyamide)-2′-deoxyuridine (2-NapdU),5-(N-naphthylmethylcarboxyamide)-2′-O-methyluridine,5-(N-naphthylmethylcarboxyamide)-2′-fluorouridine, a5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine and acombination thereof. In a related aspect, the second C-5 pyrimidinemodification scheme comprises a C-5 modified pyrimidine selected fromthe group consisting of 5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU),5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-[N-(1-naphthylmethyl)carboxyamide]-2′-deoxyuridine (NapdU),5-[N-(2-naphthylmethyl)carboxyamide]-2′-deoxyuridine (2-NapdU), and acombination thereof. In yet another related aspect, the second C-5pyrimidine modification scheme comprises a C-5 modified pyrimidineselected from the group consisting of5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-[N-(1-naphthylmethyl)carboxyamide]-2′-deoxyuridine (NapdU),5-[N-(2-naphthylmethyl)carboxyamide]-2′-deoxyuridine (2-NapdU), and acombination thereof.

In another aspect, each uracil or thymine of the second aptamer is a C-5modified pyrimidine selected from the group consisting of5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU),5-(N-benzylcarboxyamide)-2′-O-methyluridine,5-(N-benzylcarboxyamide)-2′-fluorouridine,5-(N-phenethylcarboxyamide)-2′-deoxyuridine (PEdU),5-(N-thiophenylmethylcarboxyamide)-2′-deoxyuridine (ThdU),5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),5-(N-isobutylcarboxyamide)-2′-O-methyluridine,5-(N-isobutylcarboxyamide)-2′-fluorouridine,5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-(N-tryptaminocarboxyamide)-2′-O-methyluridine,5-(N-tryptaminocarboxyamide)-2′-fluorouridine,5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2′-deoxyuridinechloride, 5-[N-(1-naphthylmethyl)carboxyamide]-2′-deoxyuridine (NapdU),5-[N-(2-naphthylmethyl)carboxyamide]-2′-deoxyuridine (2-NapdU),5-(N-naphthylmethylcarboxyamide)-2′-O-methyluridine,5-(N-naphthylmethylcarboxyamide)-2′-fluorouridine and a5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine. In arelated aspect, each uracil or thymine of the second aptamer is a5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU).

In another aspect, the percent GC content of the first aptamer andsecond aptamer are, independently, from about 37% to about 58% (or about37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%,51%, 52%, 53%, 54%, 55%, 56%, 57% or 58%).

In another aspect, the first aptamer comprises from about 9 to about 16(or about 9, 10, 11, 12, 13, 14, 15, or 16) C-5 modified pyrimidines.

In another aspect, the second aptamer comprises from about 5 to about 15C-modified pyrimidines (or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or15).

In another aspect, the first aptamer and the second aptamer,independently, are each from about 20 to 100 nucleotides in length (orfrom 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 nucleotides in length). In arelated aspect, the first aptamer and the second aptamer, independently,are from about 40 to about 100 nucleotides in length (or from 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99 or 100 nucleotides in length).

In another aspect, the first aptamer and/or the second aptamer furthercomprise a detectable moiety. In a related aspect, the detectable moietyis selected from the group consisting of a dye, a quantum dot, aradiolabel, an electrochemical functional group, an enzyme, an enzymesubstrate, a ligand and a receptor.

In another aspect, the target comprises a protein or a peptide. In arelated aspect, the target is a protein selected from the groupconsisting ANGPT2, TSP2, CRDL1, MATN2, GPVI, ESAM, C7, PLG, MMP-12,NPS-PLA2 and CdtA.

In another aspect, the dissociation constant (K_(d)) for the ternarycomplex is at least 0.02 nM, or from about 0.01 nM to about 10 nM, orfrom about 0.02 nM to about 6 nM (or from about 0.02, 0.04, 0.06, 0.08,0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1,1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5,2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4,4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5,5.6, 5.7, 5.8, 5.9 or 6 nM) or from about 0.02 nM to about 3 nM (or from0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.12, 0.14, 0.16,0.18, 0.2, 0.22, 0.24, 0.26, 0.28, 0.3, 0.32, 0.34, 0.36, 0.38, 0.4,0.42, 0.44, 0.46, 0.48, 0.5, 0.52, 0.54, 0.56, 0.58, 0.6, 0.62, 0.64,0.66, 0.68, 0.7, 0.72, 0.74, 0.76, 0.78, 0.8, 0.82, 0.84, 0.86, 0.88,0.9, 0.92, 0.94, 0.96, 0.98, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3 nM).

The present disclosure further describes a method for detecting a targetin a sample, the method comprising: contacting the sample with a firstaptamer to form a mixture, wherein the first aptamer is capable ofbinding to the target to form a first complex; incubating the mixtureunder conditions that allow for the first complex to form; contactingthe mixture with a second aptamer, wherein the second aptamer is capableof binding the first complex to form a second complex; incubating themixture under conditions that allow for the second complex to form;detecting for the presence or absence of the first aptamer, the secondaptamer, the target, the first complex or the second complex in themixture, wherein the presence of the first aptamer, the second aptamer,the target, the first complex or the second complex indicates that thetarget is present in the sample; and wherein, the first aptamercomprises a first C-5 pyrimidine modification scheme, the second aptamercomprises a second C-5 pyrimidine modification scheme, and wherein thefirst C-5 pyrimidine modification scheme and the second C-5 pyrimidinemodification scheme are different.

In one aspect, the present disclosure further provides that any of themethods disclosed herein may optionally be subject to or comprise akinetic challenge. In another aspect, the methods described hereinfurther comprise the addition of a competitor molecule, a dilution stepor one or more washes to improve the binding affinity of the aptamerwith the target. In a related aspect, the competitor molecule isselected from the group consisting of an oligonucleotide, heparin,herring sperm DNA, salmon sperm DNA, dextran sulfate, polyanion, abasicphosphodiester polymer, dNTP, and pyrophosphate. In another aspect, thekinetic challenge comprises diluting the mixture containing any of thecomplexes as described herein, and incubating the mixture containing theaptamer affinity complex for a time selected from the group consistingof greater than or equal to 30 seconds, 1 minute, 2 minutes, 3 minutes,4 minutes, 5 minutes, 10 minutes, 30 minutes, and 60 minutes. In anotheraspect, the kinetic challenge comprises diluting the mixture containingthe aptamer affinity complex and incubating the mixture containing theaptamer affinity complex for a time such that the ratio of the measuredlevel of aptamer affinity complex to the measured level of thenon-specific complex is increased.

In another aspect, the method for detecting a target in a samplecomprises contacting the sample with the first and second aptamerssimultaneously, incubating the mixture under conditions that allow theformation of a complex comprising the target and first and secondaptamers, and detecting for the presence or absence of the firstaptamer, the second aptamer, the target or the complex in the mixture,wherein the presence of the first aptamer, the second aptamer or thecomplex indicates that the target is present in the sample.

In another aspect, the first and second aptamers can both independentlyform a complex with the target. Specifically, the second aptamer canform a complex with the target alone as well as with the complex betweenthe first aptamer and the target.

In another aspect, the first aptamer has binding affinity for the targetand not the second aptamer.

In another aspect, the second aptamer has binding affinity for thetarget and not the first aptamer.

In another aspect, the second aptamer has binding affinity for the firstcomplex.

In another aspect, the first aptamer binding region of the target andthe second aptamer binding region of the target are different regions.In a related aspect, the first aptamer and the second aptamer havenon-competing binding sites on the target.

In another aspect, the first aptamer and the second aptamer,independently, comprise RNA, DNA or a combination thereof.

In another aspect, the first C-5 pyrimidine modification schemecomprises a C-5 modified pyrimidine selected from the group consistingof 5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU),5-(N-benzylcarboxyamide)-2′-O-methyluridine,5-(N-benzylcarboxyamide)-2′-fluorouridine,5-(N-phenethylcarboxyamide)-2′-deoxyuridine (PEdU),5-(N-thiophenylmethylcarboxyamide)-2′-deoxyuridine (ThdU),5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),5-(N-isobutylcarboxyamide)-2′-O-methyluridine,5-(N-isobutylcarboxyamide)-2′-fluorouridine,5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-(N-tryptaminocarboxyamide)-2′-O-methyluridine,5-(N-tryptaminocarboxyamide)-2′-fluorouridine,5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2′-deoxyuridinechloride, 5-[N-(1-naphthylmethyl)carboxyamide]-2′-deoxyuridine (NapdU),5-[N-(2-naphthylmethyl)carboxyamide]-2′-deoxyuridine (2-NapdU),5-(N-naphthylmethylcarboxyamide)-2′-O-methyluridine,5-(N-naphthylmethylcarboxyamide)-2′-fluorouridine, a5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine and acombination thereof. In a related aspect, the first C-5 pyrimidinemodification scheme comprises a C-5 modified pyrimidine selected fromthe group consisting of a 5-(N-benzylcarboxyamide)-2′-deoxyuridine(BndU), 5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-[N-(1-naphthylmethyl)carboxyamide]-2′-deoxyuridine (NapdU),5-[N-(2-naphthylmethyl)carboxyamide]-2′-deoxyuridine (2-NapdU), and acombination thereof. In yet another related aspect, the first C-5pyrimidine modification scheme comprises a C-5 modified pyrimidineselected from the group consisting of a5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU), a5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU) and a combinationthereof. In another related aspect, the first C-5 pyrimidinemodification scheme comprises a 5-(N-benzylcarboxyamide)-2′-deoxyuridine(BndU).

In another aspect, each uracil or thymine of the first aptamer is a C-5modified pyrimidine selected from the group consisting of5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU),5-(N-benzylcarboxyamide)-2′-O-methyluridine,5-(N-benzylcarboxyamide)-2′-fluorouridine,5-(N-phenethylcarboxyamide)-2′-deoxyuridine (PEdU),5-(N-thiophenylmethylcarboxyamide)-2′-deoxyuridine (ThdU),5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),5-(N-isobutylcarboxyamide)-2′-O-methyluridine,5-(N-isobutylcarboxyamide)-2′-fluorouridine,5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-(N-tryptaminocarboxyamide)-2′-O-methyluridine,5-(N-tryptaminocarboxyamide)-2′-fluorouridine,5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2′-deoxyuridinechloride5-[N-(1-naphthylmethyl)carboxyamide]-2′-deoxyuridine (NapdU),5-[N-(2-naphthylmethyl)carboxyamide]-2′-deoxyuridine (2-NapdU),5-(N-naphthylmethylcarboxyamide)-2′-O-methyluridine,5-(N-naphthylmethylcarboxyamide)-2′-fluorouridine and a5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine. In arelated aspect, each uracil or thymine of the first aptamer is a5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU).

In another aspect, the second C-5 pyrimidine modification schemecomprises a C-5 modified pyrimidine selected from the group consistingof 5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU),5-(N-benzylcarboxyamide)-2′-O-methyluridine,5-(N-benzylcarboxyamide)-2′-fluorouridine,5-(N-phenethylcarboxyamide)-2′-deoxyuridine (PEdU),5-(N-thiophenylmethylcarboxyamide)-2′-deoxyuridine (ThdU),5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),5-(N-isobutylcarboxyamide)-2′-O-methyluridine,5-(N-isobutylcarboxyamide)-2′-fluorouridine,5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-(N-tryptaminocarboxyamide)-2′-O-methyluridine,5-(N-tryptaminocarboxyamide)-2′-fluorouridine,5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2′-deoxyuridinechloride, 5-[N-(1-naphthylmethyl)carboxyamide]-2′-deoxyuridine (NapdU),5-[N-(2-naphthylmethyl)carboxyamide]-2′-deoxyuridine (2-NapdU),5-(N-naphthylmethylcarboxyamide)-2′-O-methyluridine,5-(N-naphthylmethylcarboxyamide)-2′-fluorouridine, a5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine and acombination thereof. In a related aspect, the second C-5 pyrimidinemodification scheme comprises a C-5 modified pyrimidine selected fromthe group consisting of 5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU),5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-[N-(1-naphthylmethyl)carboxyamide]-2′-deoxyuridine (NapdU),5-[N-(2-naphthylmethyl)carboxyamide]-2′-deoxyuridine (2-NapdU), and acombination thereof. In yet another related aspect, the second C-5pyrimidine modification scheme comprises a C-5 modified pyrimidineselected from the group consisting of5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-[N-(1-naphthylmethyl)carboxyamide]-2′-deoxyuridine (NapdU),5-[N-(2-naphthylmethyl)carboxyamide]-2′-deoxyuridine (2-NapdU), and acombination thereof.

In another aspect, each uracil or thymine of the second aptamer is a C-5modified pyrimidine selected from the group consisting of5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU),5-(N-benzylcarboxyamide)-2′-O-methyluridine,5-(N-benzylcarboxyamide)-2′-fluorouridine,5-(N-phenethylcarboxyamide)-2′-deoxyuridine (PEdU),5-(N-thiophenylmethylcarboxyamide)-2′-deoxyuridine (ThdU),5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),5-(N-isobutylcarboxyamide)-2′-O-methyluridine,5-(N-isobutylcarboxyamide)-2′-fluorouridine,5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-(N-tryptaminocarboxyamide)-2′-O-methyluridine,5-(N-tryptaminocarboxyamide)-2′-fluorouridine,5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2′-deoxyuridinechloride, 5-[N-(1-naphthylmethyl)carboxyamide]-2′-deoxyuridine (NapdU),5-[N-(2-naphthylmethyl)carboxyamide]-2′-deoxyuridine (2-NapdU),5-(N-naphthylmethylcarboxyamide)-2′-O-methyluridine,5-(N-naphthylmethylcarboxyamide)-2′-fluorouridine and a5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine. In arelated aspect, each uracil or thymine of the second aptamer is a5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU).

In another aspect, the first aptamer and the second aptamer,independently, are each from 20 to 100 nucleotides in length (or from20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99 or 100 nucleotides in length). In arelated aspect, the first aptamer and the second aptamer, independently,are from about 40 to about 100 nucleotides in length (or from 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99 or 100 nucleotides in length).

In another aspect, the first aptamer and/or the second aptamer furthercomprise a detectable moiety. In a related aspect, the detectable moietyis selected from the group consisting of a dye, a quantum dot, aradiolabel, an electrochemical functional group, an enzyme, an enzymesubstrate, a ligand and a receptor.

In another aspect, the target comprises a protein or a peptide. In arelated aspect, the target is a protein selected from the groupconsisting ANGPT2, TSP2, CRDL1, MATN2, GPVI, ESAM, C7, PLG, MMP-12,NPS-PLA2 and CdtA.

In another aspect, the dissociation constant (K_(d)) for the secondcomplex is at least 0.02 nM, or from about 0.01 nM to about 10 nM, orfrom about 0.02 nM to about 6 nM (or from about 0.02, 0.04, 0.06, 0.08,0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1,1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5,2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4,4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5,5.6, 5.7, 5.8, 5.9 or 6 nM) or from about 0.02 nM to about 3 nM (or from0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.12, 0.14, 0.16,0.18, 0.2, 0.22, 0.24, 0.26, 0.28, 0.3, 0.32, 0.34, 0.36, 0.38, 0.4,0.42, 0.44, 0.46, 0.48, 0.5, 0.52, 0.54, 0.56, 0.58, 0.6, 0.62, 0.64,0.66, 0.68, 0.7, 0.72, 0.74, 0.76, 0.78, 0.8, 0.82, 0.84, 0.86, 0.88,0.9, 0.92, 0.94, 0.96, 0.98, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3 nM).

In another aspect, the dissociation constant (K_(d)) for the firstcomplex is from about 0.04 nM to about 5 nM (or from 0.04, 0.06, 0.08,0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1,1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5,2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4,4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5 nM), or from about 0.04nM to about 4.8 nM (or from 0.04, 0.06, 0.08, 0.1, 0.15, 0.2, 0.25, 0.3,0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3,3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5,4.6, 4.7 or 4.8).

In another aspect, the dissociation constant (K_(d)) for the secondaptamer and the target is from about 0.03 nM to about 14 nM (or from0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9,3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4,4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9,6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4,7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9,9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.2, 10.4, 10.6,10.8, 11, 11.2, 11.4, 11.6, 11.8, 12, 12.2, 12.4, 12.6, 12.8, 13, 13.2,13.4, 13.6, 13.8 or 14 nM).

The present disclosure further describes a method comprising contactinga target with a first aptamer to form a mixture, wherein the firstaptamer is capable of binding the target to form a first complex;incubating the mixture under conditions that allow for the first complexto form; contacting the mixture with a second aptamer, wherein thesecond aptamer is capable of binding the target to form a secondcomplex; incubating the mixture under conditions that allow for thesecond complex to form; detecting for the presence or absence of thefirst aptamer and the second aptamer in the mixture, wherein thepresence of both the first aptamer and second aptamer in the mixtureindicates that the binding of the first aptamer to the target and thebinding of the second aptamer to the target is non-competitive; andwherein, the first aptamer comprises a first C-5 pyrimidine modificationscheme, the second aptamer comprises a second C-5 pyrimidinemodification scheme, and wherein the first C-5 pyrimidine modificationscheme and the second C-5 pyrimidine modification scheme are different.

In another aspect, the first aptamer has binding affinity for the targetand not the second aptamer.

In another aspect, the second aptamer has binding affinity for thetarget and not the first aptamer.

In another aspect, the second aptamer has binding affinity for the firstcomplex.

In another aspect, the first aptamer binding region of the target andthe second aptamer binding region of the target are different regions.In a related aspect, the first aptamer and the second aptamer havenon-competing binding sites on the target.

In another aspect, the first aptamer and the second aptamer,independently, comprise RNA, DNA or a combination thereof.

In another aspect, the first C-5 pyrimidine modification schemecomprises a C-5 modified pyrimidine selected from the group consistingof 5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU),5-(N-benzylcarboxyamide)-2′-O-methyluridine,5-(N-benzylcarboxyamide)-2′-fluorouridine,5-(N-phenethylcarboxyamide)-2′-deoxyuridine (PEdU),5-(N-thiophenylmethylcarboxyamide)-2′-deoxyuridine (ThdU),5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),5-(N-isobutylcarboxyamide)-2′-O-methyluridine,5-(N-isobutylcarboxyamide)-2′-fluorouridine,5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-(N-tryptaminocarboxyamide)-2′-O-methyluridine,5-(N-tryptaminocarboxyamide)-2′-fluorouridine,5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2′-deoxyuridinechloride, 5-[N-(1-naphthylmethyl)carboxyamide]-2′-deoxyuridine (NapdU),5-[N-(2-naphthylmethyl)carboxyamide]-2′-deoxyuridine (2-NapdU),5-(N-naphthylmethylcarboxyamide)-2′-O-methyluridine,5-(N-naphthylmethylcarboxyamide)-2′-fluorouridine, a5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine and acombination thereof. In a related aspect, the first C-5 pyrimidinemodification scheme comprises a C-5 modified pyrimidine selected fromthe group consisting of a 5-(N-benzylcarboxyamide)-2′-deoxyuridine(BndU), 5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-[N-(1-naphthylmethyl)carboxyamide]-2′-deoxyuridine (NapdU),5-[N-(2-naphthylmethyl)carboxyamide]-2′-deoxyuridine (2-NapdU), and acombination thereof. In yet another related aspect, the first C-5pyrimidine modification scheme comprises a C-5 modified pyrimidineselected from the group consisting of a5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU), a5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU) and a combinationthereof. In another related aspect, the first C-5 pyrimidinemodification scheme comprises a 5-(N-benzylcarboxyamide)-2′-deoxyuridine(BndU).

In another aspect, each uracil or thymine of the first aptamer is a C-5modified pyrimidine selected from the group consisting of5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU),5-(N-benzylcarboxyamide)-2′-O-methyluridine,5-(N-benzylcarboxyamide)-2′-fluorouridine,5-(N-phenethylcarboxyamide)-2′-deoxyuridine (PEdU),5-(N-thiophenylmethylcarboxyamide)-2′-deoxyuridine (ThdU),5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),5-(N-isobutylcarboxyamide)-2′-O-methyluridine,5-(N-isobutylcarboxyamide)-2′-fluorouridine,5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-(N-tryptaminocarboxyamide)-2′-O-methyluridine,5-(N-tryptaminocarboxyamide)-2′-fluorouridine,5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2′-deoxyuridinechloride, 5-[N-(1-naphthylmethyl)carboxyamide]-2′-deoxyuridine (NapdU),5-[N-(2-naphthylmethyl)carboxyamide]-2′-deoxyuridine (2-NapdU),5-(N-naphthylmethylcarboxyamide)-2′-O-methyluridine,5-(N-naphthylmethylcarboxyamide)-2′-fluorouridine and a5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine. In arelated aspect, each uracil or thymine of the first aptamer is a5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU).

In another aspect, the second C-5 pyrimidine modification schemecomprises a C-5 modified pyrimidine selected from the group consistingof 5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU),5-(N-benzylcarboxyamide)-2′-O-methyluridine,5-(N-benzylcarboxyamide)-2′-fluorouridine,5-(N-phenethylcarboxyamide)-2′-deoxyuridine (PEdU),5-(N-thiophenylmethylcarboxyamide)-2′-deoxyuridine (ThdU),5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),5-(N-isobutylcarboxyamide)-2′-O-methyluridine,5-(N-isobutylcarboxyamide)-2′-fluorouridine,5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-(N-tryptaminocarboxyamide)-2′-O-methyluridine,5-(N-tryptaminocarboxyamide)-2′-fluorouridine,5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2′-deoxyuridinechloride, 5-[N-(1-naphthylmethyl)carboxyamide]-2′-deoxyuridine (NapdU),5-[N-(2-naphthylmethyl)carboxyamide]-2′-deoxyuridine (2-NapdU),5-(N-naphthylmethylcarboxyamide)-2′-O-methyluridine,5-(N-naphthylmethylcarboxyamide)-2′-fluorouridine, a5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine and acombination thereof. In a related aspect, the second C-5 pyrimidinemodification scheme comprises a C-5 modified pyrimidine selected fromthe group consisting of 5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU),5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-[N-(1-naphthylmethyl)carboxyamide]-2′-deoxyuridine (NapdU),5-[N-(2-naphthylmethyl)carboxyamide]-2′-deoxyuridine (2-NapdU), and acombination thereof. In yet another related aspect, the second C-5pyrimidine modification scheme comprises a C-5 modified pyrimidineselected from the group consisting of5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU), a5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine (NapdU) and acombination thereof.

In another aspect, each uracil or thymine of the second aptamer is a C-5modified pyrimidine selected from the group consisting of5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU),5-(N-benzylcarboxyamide)-2′-O-methyluridine,5-(N-benzylcarboxyamide)-2′-fluorouridine,5-(N-phenethylcarboxyamide)-2′-deoxyuridine (PEdU),5-(N-thiophenylmethylcarboxyamide)-2′-deoxyuridine (ThdU),5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),5-(N-isobutylcarboxyamide)-2′-O-methyluridine,5-(N-isobutylcarboxyamide)-2′-fluorouridine,5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-(N-tryptaminocarboxyamide)-2′-O-methyluridine,5-(N-tryptaminocarboxyamide)-2′-fluorouridine,5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2′-deoxyuridinechloride, 5-[N-(1-naphthylmethyl)carboxyamide]-2′-deoxyuridine (NapdU),5-[N-(2-naphthylmethyl)carboxyamide]-2′-deoxyuridine (2-NapdU),5-(N-naphthylmethylcarboxyamide)-2′-O-methyluridine,5-(N-naphthylmethylcarboxyamide)-2′-fluorouridine and a5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine. In arelated aspect, each uracil or thymine of the second aptamer is a5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU).

In another aspect, the first aptamer and the second aptamer,independently, are each from 20 to 100 nucleotides in length (or from20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99 or 100 nucleotides in length). In arelated aspect, the first aptamer and the second aptamer, independently,are from about 40 to about 100 nucleotides in length (or from 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99 or 100 nucleotides in length).

In another aspect, the first aptamer and/or the second aptamer furthercomprise a detectable moiety. In a related aspect, the detectable moietyis selected from the group consisting of a dye, a quantum dot, aradiolabel, an electrochemical functional group, an enzyme, an enzymesubstrate, a ligand and a receptor.

In another aspect, the target comprises a protein or a peptide. In arelated aspect, the target is a protein selected from the groupconsisting ANGPT2, TSP2, CRDL1, MATN2, GPVI, ESAM, C7, PLG, MMP-12,NPS-PLA2 and CdtA.

In another aspect, the dissociation constant (K_(d)) for the secondcomplex is at least 0.02 nM, or from about 0.01 nM to about 10 nM, orfrom about 0.02 nM to about 6 nM (or from about 0.02, 0.04, 0.06, 0.08,0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1,1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5,2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4,4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5,5.6, 5.7, 5.8, 5.9 or 6 nM) or from about 0.02 nM to about 3 nM (or from0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.12, 0.14, 0.16,0.18, 0.2, 0.22, 0.24, 0.26, 0.28, 0.3, 0.32, 0.34, 0.36, 0.38, 0.4,0.42, 0.44, 0.46, 0.48, 0.5, 0.52, 0.54, 0.56, 0.58, 0.6, 0.62, 0.64,0.66, 0.68, 0.7, 0.72, 0.74, 0.76, 0.78, 0.8, 0.82, 0.84, 0.86, 0.88,0.9, 0.92, 0.94, 0.96, 0.98, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3 nM).

In another aspect, the dissociation constant (K_(d)) for the firstcomplex is from about 0.04 nM to about 5 nM (or from 0.04, 0.06, 0.08,0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1,1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5,2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4,4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5 nM), or from about 0.04nM to about 4.8 nM (or from 0.04, 0.06, 0.08, 0.1, 0.15, 0.2, 0.25, 0.3,0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3,3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5,4.6, 4.7 or 4.8).

In another aspect, the dissociation constant (K_(d)) for the secondaptamer and the target is from about 0.03 nM to about 14 nM (or from0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9,3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4,4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9,6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4,7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9,9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.2, 10.4, 10.6,10.8, 11, 11.2, 11.4, 11.6, 11.8, 12, 12.2, 12.4, 12.6, 12.8, 13, 13.2,13.4, 13.6, 13.8 or 14 nM).

The present disclosure further provides a composition comprising a firstaptamer and/or a second aptamer and a target protein, wherein the firstaptamer and/or a second aptamer and the target protein are bound by anon-covalent interaction.

The foregoing and other objects, features, and advantages of thedisclosure will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts the general strategy for the isolation and validation ofa SOMAmer (modified DNA aptamer) pair for a protein target. FIG. 1Ashows a free target used in a first SELEX (or 1st SELEX) with a modifiedrandom ssDNA library to isolate a set of aptamers, having 5′ and 3′fixed regions that can be screened directly for pairs of non-competingclones. Representative chemical modifications that may be used withinthe 40 nucleotide random region of each aptamer are provided (e.g.,abbreviated as Bn, Nap, Trp, PE, Try and 2Nap). If no pairs are present,the aptamer with the best binding properties is allowed to form acomplex with the target, which is then used in a second SELEX (or 2ndSELEX) with a different modified library. The new aptamer clones arethen screened for paired sandwich binding to the target. FIG. 1B shows arepresentation of sequence patterns and multicopy sequences selectedwith free CdtA protein (pool 5551) or with CdtA-4758-6 complex (pool5579); T=2NapdU. Non-competitive binding with the first CdtA aptamer,4758-6, is indicated by shading. FIG. 1C shows equilibrium binding ofaptamers with CdtA protein. Clones 5551-81 and 5574-49 were obtained inSELEX with free CdtA, 5579-7 through 5579-21 with CdtA-4758-6 complex ina second SELEX. The maximum bound fraction (binding plateau) in thisassay is influenced by the retention efficiency of the target-aptamercomplexes on Zorbax and the fraction of binding-competent aptamers.FIGS. 1D and 1E show a CdtA binding assay with radiolabeled 5579-12(FIG. 1D) or 5579-11 (FIG. 1E) in the absence or presence of a 100-foldexcess (10 nM) unlabeled competitor aptamer 4758-6 that was previouslygenerated for CdtA. Binding was also measured using CdtA andbiotinylated 4758-6 as a capture agent that had been pre-immobilized onstreptavidin beads. FIG. 1F shows screening for aptamer pairs on theLuminex platform using 4758-6 as capture agent bound to LumAvidin beads,and individual aptamers as detection agents. All aptamers were madesynthetically as 50-mers and contained a single biotin at their 5′-endto allow their immobilization on the beads, which were then blocked fromfurther binding with 1 mM biotin, and to allow detection with astreptavidin-phycoerythrin conjugate. FIG. 1G shows CdtA sandwich assaywith 4758-6 (25 nM) as capture agent and 5579-12 (10 nM) as detectionagent, or switching the two reagents.

FIG. 2A shows an equilibrium binding assay to screen for individualaptamer sandwich pairs, using biotinylated capture aptamers onstreptavidin beads and radiolabeled detection aptamers. FIG. 2B shows amultiplexed sandwich screening assay on the Luminex platform todistinguish competitive from non-competitive binding. Each aptamer wasimmobilized on a different LumAvidin bead type, and capture beads werepooled for testing individual aptamers as detection agents. FIG. 2Cshows pairwise screening of 16 CRDL1 aptamers in the Luminex-basedmultiplexed assay, with performance expressed as percent of maximumsignal and displayed as heat map. FIGS. 2D and 2E show the evaluation of16 CRDL1 aptamers as capture agents or detection agents) in the Luminexsandwich screening assay with SOMAmer 3362-61 which was the sequenceused to form the complex with CRDL1 during SELEX (FIG. 2D) or withSOMAmer 7575-2 which was one of the new sequences (FIG. 2E). Controlsincluded assays where the same SOMAmer was used for capture anddetection (underlined). FIG. 2F shows sandwich binding curves obtainedin the Luminex assay for proteins spiked in buffer (aptamers listed inTable 28). In all cases, the sequence that had served to form thecomplex with the target during the sandwich SELEX was used as thecapture agent, and one of the new clones identified as a result of SELEXwas used as the detection agent. The maximum signals (RFU at B_(max))were 23046 (ANGPT2), 16623 (TSP2), 23349 (CRDL1), 25586 (MATN2), 26000(C7), 13927 (GPVI), 7103 (PLG), and 3000 (ESAM), respectively.

FIG. 3 shows the K_(d) values for aptamer pairs used in a plate-basedsandwich assay. Biotinylated aptamers were immobilized onstreptavidin-coated plates as capture reagents, and used as detectionagents for labelling with streptavidin-HRP conjugate.

DETAILED DESCRIPTION I. Terms and Methods

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Aptamer: The term aptamer, as used herein, refers to a non-naturallyoccurring nucleic acid that has a desirable action on a target molecule.A desirable action includes, but is not limited to, binding of thetarget, catalytically changing the target, reacting with the target in away that modifies or alters the target or the functional activity of thetarget, covalently attaching to the target (as in a suicide inhibitor),and facilitating the reaction between the target and another molecule.

Analog: The term analog, as used herein, refers to a structural chemicalanalog as well as a functional chemical analog. A structural chemicalanalog is a compound having a similar structure to another chemicalcompound but differing by one or more atoms or functional groups. Thisdifference may be a result of the addition of atoms or functionalgroups, absence of atoms or functional groups, the replacement of atomsor functional groups or a combination thereof. A functional chemicalanalog is a compound that has similar chemical, biochemical and/orpharmacological properties. The term analog may also encompass S and Rstereoisomers of a compound.

Bioactivity: The term bioactivity, as used herein, refers to one or moreintercellular, intracellular or extracellular process (e.g., cell-cellbinding, ligand-receptor binding, cell signaling, etc.) which can impactphysiological or pathophysiological processes.

C-5 Modified Pyrimidine: C-5 modified pyrimidine, as used herein, refersto a pyrimidine with a modification at the C-5 position. Examples of aC-5 modified pyrimidine include those described in U.S. Pat. Nos.5,719,273, 5,945,527, 7,947,447, as well as, U.S. Publication No.2014/0058076, filed Feb. 27, 2014. Additional examples are providedherein.

Competitor Molecule: Competitor molecule or competitor, are usedinterchangeably to refer to any molecule that can form a non-specificcomplex with a non-target molecule. A “competitor molecule” or“competitor” is a population of different types of molecules or aparticular or species of molecule. “Competitor molecules” or“competitors” refer to more than one such type of molecules. Competitormolecules include oligonucleotides, polyanions (e.g., heparin,single-stranded salmon sperm DNA, and polydextrans (e.g., dextransulphate)), abasic phosphodiester polymers, dNTPs, and pyrophosphate. Inthe case of a kinetic challenge that uses a competitor, the competitorcan also be any molecule that can form a non-specific complex with anaptamer. Such competitor molecules include polycations (e.g., spermine,spermidine, polylysine, and polyarginine) and amino acids (e.g.,arginine and lysine).

Consensus Sequence: Consensus sequence, as used herein, refers to anucleotide sequence that represents the most frequently observednucleotide found at each position of a series of nucleic acid sequencessubject to a sequence alignment.

Covalent Bond: Covalent bond or interaction refers to a chemical bondthat involves the sharing of at least a pair of electrons between atoms.

Incubating: The term incubating (or incubation), as used herein, refersto controlled conditions in which components are placed together topromote a desired outcome. For example, a target (e.g., protein) and anaptamer may be incubated by putting them together to promote the bindingof the aptamer with the target to form a complex. Further examplesinclude putting the complex together with a second aptamer to incubatethe complex and second aptamer for form a second complex (i.e.,aptamer-protein-second aptamer). The controlled conditions forincubating include temperature, time, pH, salt concentration, and thetype of mixture, of which non-limiting examples include a solution, anemulsion, a gel and a foam. Temperatures may range from about 21° C. toabout 45° C. (or about 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 degrees celcius).Preferably, the temperature is from about 28° C. to about 37° C. (or 28,29, 30, 31, 32, 33, 34, 35, 36 or 37 degrees celcius), or about 28° C.or about 37° C. The time of incubation may include from about 1 minuteto about 240 minutes (or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170,175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235 or 240minutes). Preferably, the incubation time is about 15 minutes, 30minutes, 60 minutes, 120 minutes, 180 minutes or about 210 minutes. ThepH conditions for incubation may range from about 5 to about 12 (or 5,5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5 or 12).Preferably, the pH is about 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8,6.9, 7, 9. 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2,10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3, 11.4 or11.5). The above set of specific conditions are representative andnon-limiting. Further, the same specific conditions for incubation maybe used throughout the methods disclosed herein or may change atdifferent steps of the methods disclosed herein.

Inhibit: The term inhibit, as used herein, means to prevent or reducethe expression of a peptide or a polypeptide to an extent that thepeptide or polypeptide no longer has measurable activity or bioactivity;or to reduce the stability and/or reduce or prevent the activity of apeptide or a polypeptide to an extent that the peptide or polypeptide nolonger has measurable activity or bioactivity.

Kinetic Challenge: Kinetic challenge, as used herein, refers to aprocess of enrichment for an aptamer affinity complex from a set ofcomplexes that includes an aptamer affinity complex and non-specificcomplexes, by applying kinetic pressure and making use of the differentaffinity characteristics of the constituents of such classes ofcomplexes, including dissociation rates. A kinetic challenge generallyresults in an increase in specificity, since aptamer-non-targetcomplexes are typically reduced compared to aptamer-target complexes. Asused herein, the term “kinetic pressure” refers to a means for providingan opportunity for the natural dissociation of complexes and/orinhibiting the rebinding of molecules that dissociate from a complexnaturally. Kinetic pressure can be applied by the addition of acompetitor molecule, or by sample dilution, or by extensive washes whencomplexes are bound to a solid support, or by any other means known toone skilled in the art. As one of ordinary skill in the art willappreciate, because a kinetic challenge generally depends upon thediffering dissociation rates of aptamer affinity complexes andaptamer-non-target complexes, the duration of the kinetic challenge ischosen so as to retain a high proportion of aptamer affinity complexeswhile substantially reducing the number of aptamer-non-target complexes.For a kinetic challenge to be effective, the dissociation rate for theaptamer affinity complex is preferably significantly lower than thosefor aptamer-non-target complexes. Since an aptamer can be selected toinclude particular properties, the constituents of an aptamer affinitycomplex can be designed to have a comparatively low dissociation rate,i.e., slow off rate.

Modified: The term modified (or modify or modification) and anyvariations thereof, when used in reference to an oligonucleotide, meansthat at least one of the four constituent nucleotide bases (i.e., A, G,T/U, and C) of the oligonucleotide is an analog or ester of a naturallyoccurring nucleotide.

Modulate: The term modulate, as used herein, means to alter theexpression level of a peptide, protein or polypeptide by increasing ordecreasing its expression level relative to a reference expressionlevel, and/or alter the stability and/or activity of a peptide, proteinor polypeptide by increasing or decreasing its stability and/or activitylevel relative to a reference stability and/or activity level.

Non-covalent Bond: Non-covalent bond or non-covalent interaction refersto a chemical bond or interaction that does not involve the sharing ofpairs of electrons between atoms. Examples of non-covalent bonds orinteractions include hydrogen bonds, ionic bonds (electrostatic bonds),van der Waals forces and hydrophobic interactions.

Nucleic Acid: Nucleic acid, as used herein, refers to any nucleic acidsequence containing DNA, RNA and/or analogs thereof and may includesingle, double and multi-stranded forms. The terms “nucleic acid”,“oligo”, “oligonucleotide” and “polynucleotide” may be usedinterchangeably.

Pharmaceutically Acceptable: Pharmaceutically acceptable, as usedherein, means approved by a regulatory agency of a federal or a stategovernment or listed in the U.S. Pharmacopoeia or other generallyrecognized pharmacopoeia for use in animals and, more particularly, inhumans.

Pharmaceutically Acceptable Salt: Pharmaceutically acceptable salt orsalt of a compound (e.g., aptamer), as used herein, refers to a productthat contains an ionic bond and is typically produced by reacting thecompound with either an acid or a base, suitable for administering to anindividual. A pharmaceutically acceptable salt can include, but is notlimited to, acid addition salts including hydrochlorides, hydrobromides,phosphates, sulphates, hydrogen sulphates, alkylsulphonates,arylsulphonates, arylalkylsulfonates, acetates, benzoates, citrates,maleates, fumarates, succinates, lactates, and tartrates; alkali metalcations such as Li, Na, K, alkali earth metal salts such as Mg or Ca, ororganic amine salts.

Pharmaceutical Composition: Pharmaceutical composition, as used herein,refers to formulation comprising an aptamer in a form suitable foradministration to an individual. A pharmaceutical composition istypically formulated to be compatible with its intended route ofadministration. Examples of routes of administration include, but arenot limited to, oral and parenteral, e.g., intravenous, intradermal,subcutaneous, inhalation, topical, transdermal, transmucosal, and rectaladministration.

SELEX: The term SELEX, as used herein, refers to generally to theselection for nucleic acids that interact with a target molecule in adesirable manner, for example binding with high affinity to a protein;and the amplification of those selected nucleic acids. SELEX may be usedto identify aptamers with high affinity to a specific target molecule.The term SELEX and “SELEX process” may be used interchangeably.

Sequence Identity: Sequence identity, as used herein, in the context oftwo or more nucleic acid sequences is a function of the number ofidentical nucleotide positions shared by the sequences (i.e., %identity=number of identical positions/total number of positions×100),taking into account the number of gaps, and the length of each gap thatneeds to be introduced to optimize alignment of two or more sequences.The comparison of sequences and determination of percent identitybetween two or more sequences can be accomplished using a mathematicalalgorithm, such as BLAST and Gapped BLAST programs at their defaultparameters (e.g., Altschul et al., J. Mol. Biol. 215:403, 1990; see alsoBLASTN at www.ncbi.nlm.nih.gov/BLAST). For sequence comparisons,typically one sequence acts as a reference sequence to which testsequences are compared. When using a sequence comparison algorithm, testand reference sequences are input into a computer, subsequencecoordinates are designated if necessary, and sequence algorithm programparameters are designated. The sequence comparison algorithm thencalculates the percent sequence identity for the test sequence(s)relative to the reference sequence, based on the designated programparameters. Optimal alignment of sequences for comparison can beconducted, e.g., by the local homology algorithm of Smith and Waterman,Adv. Appl. Math., 2:482, 1981, by the homology alignment algorithm ofNeedleman and Wunsch, J. Mol. Biol., 48:443, 1970, by the search forsimilarity method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA85:2444, 1988, by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visualinspection (see generally, Ausubel, F. M. et al., Current Protocols inMolecular Biology, pub. by Greene Publishing Assoc. andWiley-Interscience (1987)). As used herein, when describing the percentidentity of a nucleic acid, the sequence of which is at least, forexample, about 95% identical to a reference nucleotide sequence, it isintended that the nucleic acid sequence is identical to the referencesequence except that the nucleic acid sequence may include up to fivepoint mutations per each 100 nucleotides of the reference nucleic acidsequence. In other words, to obtain a desired nucleic acid sequence, thesequence of which is at least about 95% identical to a reference nucleicacid sequence, up to 5% of the nucleotides in the reference sequence maybe deleted or substituted with another nucleotide, or some number ofnucleotides up to 5% of the total number of nucleotides in the referencesequence may be inserted into the reference sequence (referred to hereinas an insertion). These mutations of the reference sequence to generatethe desired sequence may occur at the 5′ or 3′ terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence.

SOMAmer: The term SOMAmer, as used herein, refers to an aptamer havingimproved off-rate characteristics. SOMAmers are alternatively referredto as Slow Off-Rate Modified Aptamers, and may be selected via theimproved SELEX methods described in U.S. Pat. No. 7,947,447, entitled“Method for Generating Aptamers with Improved Off-Rates”, which isincorporated by reference in its entirety. The terms aptamer and SOMAmermay be used interchangeably.

Spacer Sequence: Spacer sequence, as used herein, refers to any sequencecomprised of small molecule(s) covalently bound to the 5′-end, 3′-end orboth 5′ and 3′ ends of the nucleic acid sequence of an aptamer.Exemplary spacer sequences include, but are not limited to, polyethyleneglycols, hydrocarbon chains, and other polymers or copolymers thatprovide a molecular covalent scaffold connecting the consensus regionswhile preserving aptamer binding activity. In certain aspects, thespacer sequence may be covalently attached to the aptamer throughstandard linkages such as the terminal 3′ or 5′ hydroxyl, 2′ carbon, orbase modification such as the C5-position of pyrimidines, or C8 positionof purines.

Target Molecule: Target molecule (or target), as used herein, refers toany compound or molecule upon which a nucleic acid can act in adesirable manner (e.g., binding of the target, catalytically changingthe target, reacting with the target in a way that modifies or altersthe target or the functional activity of the target, covalentlyattaching to the target (as in a suicide inhibitor), and facilitatingthe reaction between the target and another molecule. Non-limitingexamples of a target molecule include a protein, peptide, nucleic acid,carbohydrate, lipid, polysaccharide, glycoprotein, hormone, receptor,antigen, antibody, virus, pathogen, toxic substance, substrate,metabolite, transition state analog, cofactor, inhibitor, drug, dye,nutrient, growth factor, cell, tissue, any portion or fragment of any ofthe foregoing, etc. Virtually any chemical or biological effector may bea suitable target. Molecules of any size can serve as targets. A targetcan also be modified in certain ways to enhance the likelihood orstrength of an interaction between the target and the nucleic acid. Atarget may also include any minor variation of a particular compound ormolecule, such as, in the case of a protein, for example, variations inits amino acid sequence, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component, which doesnot substantially alter the identity of the molecule. A “targetmolecule” or “target” is a set of copies of one type or species ofmolecule or multimolecular structure that is capable of binding to anaptamer. “Target molecules” or “targets” refer to more than one such setof molecules.

Ternary complex: Ternary complex, as used herein, refers to a complex ofat least two aptamers and a target. In certain instances, the complexmay comprise covalent, non-covalent or a combination of covalent andnon-covalent interactions.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. “Comprising A or B” means including A or B, orincluding A and B. It is further to be understood that all base sizes oramino acid sizes, and all molecular weight or molecular mass values,given for nucleic acids or polypeptides are approximate, and areprovided for description.

Further, ranges provided herein are understood to be shorthand for allof the values within the range. For example, a range of 1 to 50 isunderstood to include any number, combination of numbers, or sub-rangefrom the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or50 (as well as fractions thereof unless the context clearly dictatesotherwise). Any concentration range, percentage range, ratio range, orinteger range is to be understood to include the value of any integerwithin the recited range and, when appropriate, fractions thereof (suchas one tenth and one hundredth of an integer), unless otherwiseindicated. Also, any number range recited herein relating to anyphysical feature, such as polymer subunits, size or thickness, are to beunderstood to include any integer within the recited range, unlessotherwise indicated. As used herein, “about” or “consisting essentiallyof mean±20% of the indicated range, value, or structure, unlessotherwise indicated. As used herein, the terms “include” and “comprise”are open ended and are used synonymously. It should be understood thatthe terms “a” and “an” as used herein refer to “one or more” of theenumerated components. The use of the alternative (e.g., “or”) should beunderstood to mean either one, both, or any combination thereof of thealternatives.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present disclosure,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

II. Overview

In another aspect of this disclosure, the first aptamer and the secondaptamer of the present disclosure may include up to about 100nucleotides, up to about 95 nucleotides, up to about 90 nucleotides, upto about 85 nucleotides, up to about 80 nucleotides, up to about 75nucleotides, up to about 70 nucleotides, up to about 65 nucleotides, upto about 60 nucleotides, up to about 55 nucleotides, up to about 50nucleotides, up to about 45 nucleotides, up to about 40 nucleotides, upto about 35 nucleotides, up to about 30 nucleotides, up to about 25nucleotides, and up to about 20 nucleotides.

In another aspect of this disclosure, the first C-5 pyrimidinemodification scheme improves the off-rate or the rate of dissociation ofthe first aptamer compared to the first aptamer without the first C-5pyrimidine modification scheme. In another aspect, the second C-5pyrimidine modification scheme improves the off-rate or the rate ofdissociation of the second aptamer compared to the second aptamerwithout the second C-5 pyrimidine modification scheme.

In another aspect of this disclosure, the first aptamer may be at leastabout 95% identical, at least about 90% identical, at least about 85%identical, at least about 80% identical, or at least about 75% identicalto another nucleic acid sequence of another aptamer. In another aspectof this disclosure, the second aptamer may be at least about 95%identical, at least about 90% identical, at least about 85% identical,at least about 80% identical, or at least about 75% identical to anothernucleic acid sequence of another aptamer.

In another aspect, the K_(d) of the first or second aptamer to thetarget or target/aptamer complex is from about 1 nM to about 100 nM (orfrom 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 90, 91,92, 93, 94, 95, 96, 97, 98, 99 or 100 nM).

In another aspect, the K_(d) is from about 4 nM to about 10 nM (or from4, 5, 6, 7, 8, 9, or 10 nM).

In another aspect this disclosure, the first aptamer and/or secondaptamer may have a dissociation constant (K_(d)) for the target or atarget/aptamer complex of about 10 nM or less. In another exemplaryembodiment, the first aptamer and/or second aptamer has a dissociationconstant (K_(d)) for the target protein of about 15 nM or less. In yetanother exemplary embodiment, the first aptamer and/or second aptamerhas a dissociation constant (K_(d)) for the target protein of about 20nM or less. In yet another exemplary embodiment, the first aptamerand/or second aptamer has a dissociation constant (K_(d)) for the targetprotein of about 25 nM or less. In yet another exemplary embodiment, thefirst aptamer and/or second aptamer has a dissociation constant (K_(d))for the target protein of about 30 nM or less. In yet another exemplaryembodiment, the first aptamer and/or second aptamer has a dissociationconstant (K_(d)) for the target protein of about 35 nM or less. In yetanother exemplary embodiment, the first aptamer and/or second aptamerhas a dissociation constant (K_(d)) for the target protein of about 40nM or less. In yet another exemplary embodiment, the first aptamerand/or second aptamer has a dissociation constant (K_(d)) for the targetprotein of about 45 nM or less. In yet another exemplary embodiment, thefirst aptamer and/or second aptamer has a dissociation constant (K_(d))for the target protein of about 50 nM or less. In yet another exemplaryembodiment, the first aptamer and/or second aptamer has a dissociationconstant (K_(d)) for the target protein in a range of about 3-10 nM (or3, 4, 5, 6, 7, 8, 9 or 10 nM). In yet another exemplary embodiment, thefirst aptamer and/or second aptamer has a dissociation constant (K_(d))for the target protein in a range of about 0.02 nM to about 3 nM (orfrom 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.12, 0.14,0.16, 0.18, 0.2, 0.22, 0.24, 0.26, 0.28, 0.3, 0.32, 0.34, 0.36, 0.38,0.4, 0.42, 0.44, 0.46, 0.48, 0.5, 0.52, 0.54, 0.56, 0.58, 0.6, 0.62,0.64, 0.66, 0.68, 0.7, 0.72, 0.74, 0.76, 0.78, 0.8, 0.82, 0.84, 0.86,0.88, 0.9, 0.92, 0.94, 0.96, 0.98, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3 nM).

A suitable dissociation constant can be determined with a binding assayusing a multi-point titration and fitting the equationy=(max−min)(Protein)/(K_(d)+Protein)+min as described herein. It is tobe understood that the determination of dissociation constants is highlydependent upon the conditions under which they are measured and thusthese numbers may vary significantly with respect to factors such asequilibration time, etc.

In another aspect of this disclosure, the first aptamer and/or secondaptamer comprise a rate of dissociation (t_(1/2)) from the targetselected from the group consisting of a time ≥about 15 minutes, ≥about30 minutes, ≥about 60 minutes, ≥about 90 minutes, ≥about 120 minutes,≥about 150 minutes, ≥about 180 minutes, ≥about 210 minutes and ≥about240 minutes.

The present disclosure further provides kits comprising a first aptamerand a second aptamer, wherein the first aptamer comprises a first C-5pyrimidine modification scheme, the second aptamer comprises a secondC-5 pyrimidine modification scheme, and wherein the first C-5 pyrimidinemodification scheme and the second C-5 pyrimidine modification schemeare different; and wherein the first aptamer has binding affinity for atarget, and the second aptamer has binding affinity for the targetand/or the first aptamer bound to the target.

In another aspect, the first aptamer has affinity for the target and notthe second aptamer.

In another aspect, the second aptamer has binding affinity for thetarget and not the first aptamer.

In another aspect, the second aptamer has binding affinity for a complexformed by the association of the first aptamer with the target.

In another aspect, the first aptamer binding region of the target andthe second aptamer binding region of the target are different regions.In a related aspect, the first aptamer and the second aptamer havenon-competing binding sites on the target.

In another aspect, the first aptamer and the second aptamer,independently, comprise RNA, DNA or a combination thereof.

In another aspect, the first C-5 pyrimidine modification schemecomprises a C-5 modified pyrimidine selected from the group consistingof 5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU),5-(N-benzylcarboxyamide)-2′-O-methyluridine,5-(N-benzylcarboxyamide)-2′-fluorouridine,5-(N-phenethylcarboxyamide)-2′-deoxyuridine (PEdU),5-(N-thiophenylmethylcarboxyamide)-2′-deoxyuridine (ThdU),5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),5-(N-isobutylcarboxyamide)-2′-O-methyluridine,5-(N-isobutylcarboxyamide)-2′-fluorouridine,5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-(N-tryptaminocarboxyamide)-2′-O-methyluridine,5-(N-tryptaminocarboxyamide)-2′-fluorouridine,5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2′-deoxyuridinechloride, 5-[N-(1-naphthylmethyl)carboxyamide]-2′-deoxyuridine (NapdU),5-[N-(2-naphthylmethyl)carboxyamide]-2′-deoxyuridine (2-NapdU),5-(N-naphthylmethylcarboxyamide)-2′-O-methyluridine,5-(N-naphthylmethylcarboxyamide)-2′-fluorouridine, a5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine and acombination thereof. In a related aspect, the first C-5 pyrimidinemodification scheme comprises a C-5 modified pyrimidine selected fromthe group consisting of a 5-(N-benzylcarboxyamide)-2′-deoxyuridine(BndU), 5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-[N-(1-naphthylmethyl)carboxyamide]-2′-deoxyuridine (NapdU),5-[N-(2-naphthylmethyl)carboxyamide]-2′-deoxyuridine (2-NapdU), and acombination thereof. In yet another related aspect, the first C-5pyrimidine modification scheme comprises a C-5 modified pyrimidineselected from the group consisting of a5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU), a5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU) and a combinationthereof. In another related aspect, the first C-5 pyrimidinemodification scheme comprises a 5-(N-benzylcarboxyamide)-2′-deoxyuridine(BndU).

In another aspect, each uracil or thymine of the first aptamer is a C-5modified pyrimidine selected from the group consisting of5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU),5-(N-benzylcarboxyamide)-2′-O-methyluridine,5-(N-benzylcarboxyamide)-2′-fluorouridine,5-(N-phenethylcarboxyamide)-2′-deoxyuridine (PEdU),5-(N-thiophenylmethylcarboxyamide)-2′-deoxyuridine (ThdU),5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),5-(N-isobutylcarboxyamide)-2′-O-methyluridine,5-(N-isobutylcarboxyamide)-2′-fluorouridine,5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-(N-tryptaminocarboxyamide)-2′-O-methyluridine,5-(N-tryptaminocarboxyamide)-2′-fluorouridine,5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2′-deoxyuridinechloride, 5-[N-(1-naphthylmethyl)carboxyamide]-2′-deoxyuridine (NapdU),5-[N-(2-naphthylmethyl)carboxyamide]-2′-deoxyuridine (2-NapdU),5-(N-naphthylmethylcarboxyamide)-2′-O-methyluridine,5-(N-naphthylmethylcarboxyamide)-2′-fluorouridine and a5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine. In arelated aspect, each uracil or thymine of the first aptamer is a5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU).

In another aspect, the second C-5 pyrimidine modification schemecomprises a C-5 modified pyrimidine selected from the group consistingof 5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU),5-(N-benzylcarboxyamide)-2′-O-methyluridine,5-(N-benzylcarboxyamide)-2′-fluorouridine,5-(N-phenethylcarboxyamide)-2′-deoxyuridine (PEdU),5-(N-thiophenylmethylcarboxyamide)-2′-deoxyuridine (ThdU),5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),5-(N-isobutylcarboxyamide)-2′-O-methyluridine,5-(N-isobutylcarboxyamide)-2′-fluorouridine,5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-(N-tryptaminocarboxyamide)-2′-O-methyluridine,5-(N-tryptaminocarboxyamide)-2′-fluorouridine,5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2′-deoxyuridinechloride, 5-[N-(1-naphthylmethyl)carboxyamide]-2′-deoxyuridine (NapdU),5-[N-(2-naphthylmethyl)carboxyamide]-2′-deoxyuridine (2-NapdU),5-(N-naphthylmethylcarboxyamide)-2′-O-methyluridine,5-(N-naphthylmethylcarboxyamide)-2′-fluorouridine, a5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine and acombination thereof. In a related aspect, the second C-5 pyrimidinemodification scheme comprises a C-5 modified pyrimidine selected fromthe group consisting of 5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU),5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-[N-(1-naphthylmethyl)carboxyamide]-2′-deoxyuridine (NapdU),5-[N-(2-naphthylmethyl)carboxyamide]-2′-deoxyuridine (2-NapdU), and acombination thereof. In yet another related aspect, the second C-5pyrimidine modification scheme comprises a C-5 modified pyrimidineselected from the group consisting of5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-[N-(1-naphthylmethyl)carboxyamide]-2′-deoxyuridine (NapdU),5-[N-(2-naphthylmethyl)carboxyamide]-2′-deoxyuridine (2-NapdU), and acombination thereof.

In another aspect, each uracil or thymine of the second aptamer is a C-5modified pyrimidine selected from the group consisting of5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU),5-(N-benzylcarboxyamide)-2′-O-methyluridine,5-(N-benzylcarboxyamide)-2′-fluorouridine,5-(N-phenethylcarboxyamide)-2′-deoxyuridine (PEdU),5-(N-thiophenylmethylcarboxyamide)-2′-deoxyuridine (ThdU),5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),5-(N-isobutylcarboxyamide)-2′-O-methyluridine,5-(N-isobutylcarboxyamide)-2′-fluorouridine,5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-(N-tryptaminocarboxyamide)-2′-O-methyluridine,5-(N-tryptaminocarboxyamide)-2′-fluorouridine,5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2′-deoxyuridinechloride, 5-[N-(1-naphthylmethyl)carboxyamide]-2′-deoxyuridine (NapdU),5-[N-(2-naphthylmethyl)carboxyamide]-2′-deoxyuridine (2-NapdU),5-(N-naphthylmethylcarboxyamide)-2′-O-methyluridine,5-(N-naphthylmethylcarboxyamide)-2′-fluorouridine and a5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine. In arelated aspect, each uracil or thymine of the second aptamer is a5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU).

In another aspect, the first aptamer and the second aptamer,independently, are each from about 20 to 100 nucleotides in length (orfrom 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 nucleotides in length). In arelated aspect, the first aptamer and the second aptamer, independently,are from about 40 to about 100 nucleotides in length (or from 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99 or 100 nucleotides in length).

In another aspect, the first aptamer and/or the second aptamer furthercomprise a detectable moiety. In a related aspect, the detectable moietyis selected from the group consisting of a dye, a quantum dot, aradiolabel, an electrochemical functional group, an enzyme, an enzymesubstrate, a ligand and a receptor.

In another aspect, the target comprises a protein or a peptide. In arelated aspect, the target is a protein selected from the groupconsisting ANGPT2, TSP2, CRDL1, MATN2, GPVI, ESAM, C7, PLG, MMP-12,NPS-PLA2 and CdtA.

The present disclosure further provides that any of the methodsdisclosed herein may be subject to or comprise a kinetic challenge. Inanother aspect, the methods described herein further comprise theaddition of a competitor molecule, a dilution step or one or more washesto improve the binding affinity of the aptamer with the target. In arelated aspect, the competitor molecule is selected from the groupconsisting of an oligonucleotide, heparin, herring sperm DNA, salmonsperm DNA, dextran sulfate, polyanion, abasic phosphodiester polymer,dNTP, and pyrophosphate. In another aspect, the kinetic challengecomprises diluting the mixture containing any of the complexes asdescribed herein, and incubating the mixture containing the aptameraffinity complex for a time selected from the group consisting ofgreater than or equal to 30 seconds, 1 minute, 2 minutes, 3 minutes, 4minutes, 5 minutes, 10 minutes, 30 minutes, and 60 minutes. In anotheraspect, the kinetic challenge comprises diluting the mixture containingthe aptamer affinity complex and incubating the mixture containing theaptamer affinity complex for a time such that the ratio of the measuredlevel of aptamer affinity complex to the measured level of thenon-specific complex is increased.

The present disclosure describes a composition comprising a firstaptamer, second aptamer and a target, wherein the first aptamercomprises a first C-5 pyrimidine modification scheme, the second aptamercomprises a second C-5 pyrimidine modification scheme, and wherein thefirst C-5 pyrimidine modification scheme and the second C-5 pyrimidinemodification scheme are the same and wherein the first aptamer, secondaptamer and the target are capable of forming a ternary complex.

The present disclosure further describes a method for detecting a targetin a sample, the method comprising: contacting the sample with a firstaptamer to form a mixture, wherein the first aptamer is capable ofbinding to the target to form a first complex; incubating the mixtureunder conditions that allow for the first complex to form; contactingthe mixture with a second aptamer, wherein the second aptamer is capableof binding the first complex to form a second complex; incubating themixture under conditions that allow for the second complex to form;detecting for the presence or absence of the first aptamer, the secondaptamer, the target, the first complex or the second complex in themixture, wherein the presence of the first aptamer, the second aptamer,the target, the first complex or the second complex indicates that thetarget is present in the sample; and wherein, the first aptamercomprises a first C-5 pyrimidine modification scheme, the second aptamercomprises a second C-5 pyrimidine modification scheme, and wherein thefirst C-5 pyrimidine modification scheme and the second C-5 pyrimidinemodification scheme are the same.

In another aspect, the method for detecting a target in a samplecomprises contacting the sample with the first and second aptamerssimultaneously, incubating the mixture under conditions that allow theformation of a complex comprising the target and first and secondaptamers, and detecting for the presence or absence of the firstaptamer, the second aptamer, the target or the complex in the mixture,wherein the presence of the first aptamer, the second aptamer or thecomplex indicates that the target is present in the sample.

In another aspect, the first and second aptamers can both independentlyform a complex with the target. Specifically, the second aptamer canform a complex with the target alone as well as with the complex betweenthe first aptamer and the target.

The present disclosure further describes a method comprising contactinga target with a first aptamer to form a mixture, wherein the firstaptamer is capable of binding the target to form a first complex;incubating the mixture under conditions that allow for the first complexto form; contacting the mixture with a second aptamer, wherein thesecond aptamer is capable of binding the target to form a secondcomplex; incubating the mixture under conditions that allow for thesecond complex to form; detecting for the presence or absence of thefirst aptamer and the second aptamer in the mixture, wherein thepresence of both the first aptamer and second aptamer in the mixtureindicates that the binding of the first aptamer to the target and thebinding of the second aptamer to the target is non-competitive; andwherein, the first aptamer comprises a first C-5 pyrimidine modificationscheme, the second aptamer comprises a second C-5 pyrimidinemodification scheme, and wherein the first C-5 pyrimidine modificationscheme and the second C-5 pyrimidine modification scheme are the same.

In another aspect, the first aptamer is capable of binding a proteinselected from the group consisting ANGPT2, TSP2, CRDL1, MATN2, GPVI,ESAM, C7, PLG, MMP-12, NPS-PLA2 and CdtA.

In another aspect, the second aptamer is capable of binding a proteinselected from the group consisting ANGPT2, TSP2, CRDL1, MATN2, GPVI,ESAM, C7, PLG, MMP-12, NPS-PLA2 and CdtA.

In another aspect, the first aptamer, the second aptamer and the targetform a ternary complex, wherein the first aptamer binds the target in afirst region of the target, and the second aptamer binds the target in asecond region of the target, wherein the first region and second regionof the target are overlapping or non-overlapping regions.

In another aspect, the composition comprises a first aptamer, a secondaptamer and a target, wherein the first aptamer, second aptamer and thetarget are capable of forming a ternary complex, and wherein the firstaptamer and second aptamer are, independently, from about 40 to about 50nucleotides in length, and the first aptamer comprises a C-5 modifiedpyrimidine and the second aptamer comprises a C-5 modified pyrimidine,wherein the C-5 modified pyrimidine is selected from the groupconsisting of a 5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU),5-(N-benzylcarboxyamide)-2′-O-methyluridine,5-(N-benzylcarboxyamide)-2′-fluorouridine,5-(N-phenethylcarboxyamide)-2′-deoxyuridine (PEdU),5-[N-(phenyl-3-propyl)carboxamide]-2′-deoxyuridine (PPdU),5-[N-(2-thiophene-methyl)carboxamide]-2′-deoxyuridine (ThdU) (alsoreferred to as 5-(N-thiophenylmethylcarboxyamide)-2′-deoxyuridine),5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),5-(N-isobutylcarboxyamide)-2′-O-methyluridine,5-(N-isobutylcarboxyamide)-2′-fluorouridine,5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-(N-tryptaminocarboxyamide)-2′-O-methyluridine,5-(N-tryptaminocarboxyamide)-2′-fluorouridine,5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2′-deoxyuridinechloride, 5-[N-(1-naphthylmethyl)carboxyamide]-2′-deoxyuridine (NapdU),5-[N-(2-naphthylmethyl)carboxyamide]-2′-deoxyuridine (2-NapdU),5-[N-(1-naphthylethyl)carboxyamide]-2′-deoxyuridine (NEdU),5-[N-(2-naphthylethyl)carboxyamide]-2′-deoxyuridine 2NEdU),5-[N-(4-fluorobenzyl)carboxyamide]-2′-deoxyuridine FBndU),5-[N-(4-hydroxyphenyl-2-ethyl)carboxamide]-2′-deoxyuridine (TyrdU),5-(N-naphthylmethylcarboxyamide)-2′-O-methyluridine,5-(N-naphthylmethylcarboxyamide)-2′-fluorouridine,5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine,5-[N-(3-benzo[b]thiophene-2-ethyl)carboxamide]-2′-deoxyuridine (BTdU),5-[N-(3-benzo[a]furan-2-ethyl)carboxamide]-2′-deoxyuridine (BFdU),5-[N-(3,4-methylenedioxybenzyl)carboxamide]-2′-deoxyuridine (MBndU),5-[N—((R)-2-tetrahydrofurylmethyl)carboxamide]-2′-deoxyuridine (RTHdU),5-[N—((S)-2-tetrahydrofurylmethyl)carboxamide]-2′-deoxyuridine (STHFdU),5-(N-2-imidazolylethylcarboxamide)-2′-deoxyuridine (ImiddU),5-[N-(1-morpholino-2-ethyl)carboxamide]-2′-deoxyuridine (MOEdU), andwherein the C-5 modified pyrimidine of the first aptamer and the C-5modified pyrimidine of the second aptamer are different C-5 modifiedpyrimidines.

In another aspect, the composition comprises a first aptamer, a secondaptamer and a target, wherein the first aptamer, second aptamer and thetarget are capable of forming a ternary complex, and wherein the firstaptamer and second aptamer are, independently, from about 40 to about 50nucleotides in length, and the first aptamer comprises a C-5 modifiedpyrimidine and the second aptamer comprises a C-5 modified pyrimidine,wherein the C-5 modified pyrimidine is selected from the groupconsisting of a 5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU),5-(N-benzylcarboxyamide)-2′-O-methyluridine,5-(N-benzylcarboxyamide)-2′-fluorouridine,5-(N-phenethylcarboxyamide)-2′-deoxyuridine (PEdU),5-[N-(phenyl-3-propyl)carboxamide]-2′-deoxyuridine (PPdU),5-[N-(2-thiophene-methyl)carboxamide]-2′-deoxyuridine (ThdU),5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),5-(N-isobutylcarboxyamide)-2′-O-methyluridine,5-(N-isobutylcarboxyamide)-2′-fluorouridine,5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-(N-tryptaminocarboxyamide)-2′-O-methyluridine,5-(N-tryptaminocarboxyamide)-2′-fluorouridine,5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2′-deoxyuridinechloride, 5-[N-(1-naphthylmethyl)carboxyamide]-2′-deoxyuridine (NapdU),5-[N-(2-naphthylmethyl)carboxyamide]-2′-deoxyuridine (2-NapdU),5-[N-(1-naphthylethyl)carboxyamide]-2′-deoxyuridine (NEdU),5-[N-(2-naphthylethyl)carboxyamide]-2′-deoxyuridine 2NEdU),5-[N-(4-fluorobenzyl)carboxyamide]-2′-deoxyuridine FBndU),5-[N-(4-hydroxyphenyl-2-ethyl)carboxamide]-2′-deoxyuridine (TyrdU),5-(N-naphthylmethylcarboxyamide)-2′-O-methyluridine,5-(N-naphthylmethylcarboxyamide)-2′-fluorouridine,5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine,5-[N-(3-benzo[b]thiophene-2-ethyl)carboxamide]-2′-deoxyuridine (BTdU),5-[N-(3-benzo[a]furan-2-ethyl)carboxamide]-2′-deoxyuridine (BFdU),5-[N-(3,4-methylenedioxybenzyl)carboxamide]-2′-deoxyuridine (MBndU),5-[N—((R)-2-tetrahydrofurylmethyl)carboxamide]-2′-deoxyuridine (RTHdU),5-[N—((S)-2-tetrahydrofurylmethyl)carboxamide]-2′-deoxyuridine (STHFdU),5-(N-2-imidazolylethylcarboxamide)-2′-deoxyuridine (ImiddU),5-[N-(1-morpholino-2-ethyl)carboxamide]-2′-deoxyuridine (MOEdU), andwherein the C-5 modified pyrimidine of the first aptamer and the C-5modified pyrimidine of the second aptamer are the same C-5 modifiedpyrimidines.

In another aspect, the mixture of the first aptamer and target areincubated under conditions that allow for the first complex to form.These conditions include a target (e.g., protein) to first aptamer ratioof about 10:1. Alternative ratios of target to first aptamer includefrom about 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4,1:5, 1:6, 1:7, 1:8, 1:9 and 1:10. The conditions further include atemperature of about 37° C. Alternative temperatures include roomtemperature (or about 21° C.) or from about 21° C. to about 37° C. (or21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37°C.). The conditions also include times of incubation at theaforementioned ratio and temperature conditions, including from about 30seconds to about 72 hours (or from about 30 seconds, 1 minute, 2,minutes, 5 minutes, 10, minutes, 15, minutes, 20 minutes, 30 minutes, 45minutes, 1 hour, 2 hours, 3, hours, 4 hours, 5 hours, 6 hours, 7 hours,8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 20 hours, 24 hours, 30hours, 36 hours, 40 hours, 44 hours, 48 hours, 50 hours, 55 hours, 60hours, 65 hours, 70 hours or 72 hours).

In another aspect, the mixture of the second aptamer and target or thesecond aptamer and the first complex are incubated under conditions thatallow for the second complex to form. These conditions include a target(e.g., protein) or first complex to second aptamer ratio of about 10:1.Alternative ratios of target or first complex to second aptamer includefrom about 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4,1:5, 1:6, 1:7, 1:8, 1:9 and 1:10. The conditions further include atemperature of about 37° C. Alternative temperatures include roomtemperature (or about 21° C.) or from about 21° C. to about 37° C. (or21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37°C.). The conditions also include times of incubation at theaforementioned ratio and temperature conditions, including from about 30seconds to about 72 hours (or from about 30 seconds, 1 minute, 2,minutes, 5 minutes, 10, minutes, 15, minutes, 20 minutes, 30 minutes, 45minutes, 1 hour, 2 hours, 3, hours, 4 hours, 5 hours, 6 hours, 7 hours,8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 20 hours, 24 hours, 30hours, 36 hours, 40 hours, 44 hours, 48 hours, 50 hours, 55 hours, 60hours, 65 hours, 70 hours or 72 hours).

In another aspect, the binding affinity (or K_(d)) is determined by themethods selected from a radiolabel filter-binding assay and afluorescence bead-based Luminex assay.

The present disclosure further describes a method comprising contactinga first aptamer with a solid support, wherein the first aptamercomprises a linker and a tag, wherein the tag is capable of binding tothe solid support; contacting a target with the first aptamer, whereinthe first aptamer has binding affinity for the target, and the firstaptamer binds the target to form a first complex; contacting the firstcomplex with a plurality of aptamers, wherein at least one aptamer ofthe plurality of aptamers binds the first complex to form a secondcomplex; partitioning the second complex from the remaining plurality ofaptamers;

dissociating the second complex; amplifying the at least one aptamer;and identifying the at least one aptamer that is capable of binding thefirst complex.

The present disclosure further describes a method comprising contactinga first aptamer with a solid support, wherein the first aptamercomprises a linker and a tag, wherein the tag is capable of binding tothe solid support; contacting a target with the first aptamer, whereinthe first aptamer has binding affinity for the target, and the firstaptamer binds the target to form a first complex; contacting the firstcomplex with a plurality of aptamers, wherein one or more aptamers ofthe plurality of aptamers bind the first complex to form a secondcomplex; partitioning the second complex from the remaining plurality ofaptamers;

dissociating the second complex; amplifying the one or more aptamers andidentifying the one or more aptamers that are capable of binding thefirst complex.

The present disclosure further describes a method comprising contactinga first aptamer with a solid support, wherein the first aptamercomprises a linker and a tag, wherein the tag is capable of binding tothe solid support; contacting a target with the first aptamer, whereinthe first aptamer has binding affinity for the target, and the firstaptamer binds the target to form a first complex; contacting the firstcomplex with a plurality of aptamers to form a second complex, whereinthe second complex comprises the first aptamer, the target and a secondaptamer; partitioning the second complex from the remaining plurality ofaptamers; dissociating the second complex; amplifying the second aptamerand identifying the second aptamer that is capable of binding the firstcomplex.

In another aspect, the first aptamer comprises a C-5 modifiedpyrimidine.

In another aspect, the at least one aptamer comprises a C-5 modifiedpyrimidine.

In another aspect, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%or 100% of the aptamers of the plurality of aptamers comprise a C-5modified pyrimidine.

In another aspect, the tag binds the solid support.

In another aspect, the remaining plurality of aptamers have less bindingaffinity for the first complex than the at least one aptamer.

In another aspect, the tag is at the 5′-end or the 3′-end of the firstaptamer.

In another aspect, the linker is a photo-cleavable linker.

In another aspect, the target is s selected from the group consisting ofa protein, a peptide, a carbohydrate, a glycoprotein, a cell and atissue.

In another aspect, the first aptamer comprises a first C-5 pyrimidinemodification scheme, the at least one aptamer comprises a second C-5pyrimidine modification scheme, and wherein the first C-5 pyrimidinemodification scheme and the second C-5 pyrimidine modification schemeare the same or are different.

In another aspect, the first aptamer comprises a first C-5 pyrimidinemodification scheme, the one or more aptamers comprise a second C-5pyrimidine modification scheme, and wherein the first C-5 pyrimidinemodification scheme and the second C-5 pyrimidine modification schemeare the same or are different.

In another aspect, the first aptamer comprises a first C-5 pyrimidinemodification scheme, the second aptamer comprises a second C-5pyrimidine modification scheme, and wherein the first C-5 pyrimidinemodification scheme and the second C-5 pyrimidine modification schemeare the same or are different.

In another aspect, the at least one aptamer has binding affinity for thetarget and not the first aptamer.

In another aspect, the at least one aptamer binds the target of thefirst complex and not the first aptamer of the first complex.

In another aspect, the at least one aptamer binds the target and thefirst aptamer of the first complex.

In another aspect, the one or more aptamers have binding affinity forthe target and not the first aptamer.

In another aspect, the one or more aptamers bind the target of the firstcomplex and not the first aptamer of the first complex.

In another aspect, the one or more aptamers bind the target and thefirst aptamer of the first complex.

In another aspect, the second aptamer has binding affinity for thetarget and not the first aptamer.

In another aspect, the second aptamer binds the target of the firstcomplex and not the first aptamer of the first complex.

In another aspect, the second aptamer binds the target and the firstaptamer of the first complex.

In another aspect, the solid support is selected from the groupconsisting of a microscope slide, a cyclo-olefin copolymer substrate, amembrane, a plastic substrate, a paramagnetic bead, charged paper,nylon, a Langmuir-Bodgett film, glass, a germanium substrate, a siliconsubstrate, a silicon wafer chip, a flow through chip, a microbead, apolytetrafluoroethylene substrate, a polystyrene substrate, a galliumarsenide substrate, a gold substrate and a silver substrate.

In another aspect, the solid support is a streptavidin bead.

In another aspect, the amplification step results in the formation of acandidate mixture of aptamers.

In another aspect, the method further comprises contacting the firstcomplex with the candidate mixture of aptamers to further select foraptamers with binding affinity for the first complex.

The disclosure further provides for a method comprising: a) contacting afirst aptamer with a solid support, wherein the first aptamer comprisesa linker and a tag, wherein the tag is capable of binding to the solidsupport; b) contacting a target with the first aptamer, wherein thefirst aptamer has binding affinity for the target, and the first aptamerbinds the target to form a first complex; c) contacting the firstcomplex with a plurality of aptamers to form a plurality of secondcomplexes, wherein each of the plurality of second complexes comprisesthe first aptamer, the target and a second aptamer, and wherein theplurality of second complexes comprises a plurality of second aptamers;d) partitioning the plurality of second complexes from the aptamers ofthe plurality of aptamers, wherein at least one aptamer of the pluralityof aptamers has less binding affinity for the first complex than atleast one of the second aptamers of the plurality of second complexes;e) dissociating the plurality of second complexes; f) amplifying theplurality of second aptamers to form a first candidate mixture ofaptamers; g) at least one time, repeating steps a) through f) with thefirst candidate mixture of aptamers to form a second candidate mixtureof aptamers, or at last two times, repeating steps a) through f) to forma third candidate mixture of aptamers, or at last three times, repeatingsteps a) through f) to form a fourth candidate mixture of aptamers, orat last four times, repeating steps a) through f) to form a fifthcandidate mixture of aptamers, or at last five times, repeating steps a)through f) to form a sixth candidate mixture of aptamers, or at last sixtimes, repeating steps a) through f) to form a seventh candidate mixtureof aptamers, or at last seven times, repeating steps a) through f) toform an eighth candidate mixture of aptamers, or at last eight times,repeating steps a) through f) to form a ninth candidate mixture ofaptamers, or at last nine times, repeating steps a) through f) to form atenth candidate mixture of aptamers, or at last ten times, repeatingsteps a) through f) to form a eleventh candidate mixture of aptamers, orat last eleven times, repeating steps a) through f) to form a twelfthcandidate mixture of aptamers, or at last twelve times, repeating stepsa) through f) to form a thirteenth candidate mixture of aptamers, or atlast thirteen times, repeating steps a) through f) to form a fourteenthcandidate mixture of aptamers, or at last fourteen times, repeatingsteps a) through f) to form a fifteenth candidate mixture of aptamers,or at last fifteen times, repeating steps a) through f) to form asixteenth candidate mixture of aptamers and h) identifying at least oneof the aptamers of the plurality of second aptamers that is capable ofbinding the first complex.

The disclosure further provides for a method comprising a) contacting afirst aptamer with a solid support, wherein the first aptamer comprisesa linker and a tag, wherein the tag is capable of binding to the solidsupport; b) contacting a target with the first aptamer, wherein thefirst aptamer has binding affinity for the target, and the first aptamerbinds the target to form a first complex; c) contacting the firstcomplex with a plurality of aptamers to form a plurality of secondcomplexes, wherein each of the plurality of second complexes comprisesthe first aptamer, the target and a second aptamer, and wherein theplurality of second complexes comprises a plurality of second aptamers;d) partitioning the plurality of second complexes from the aptamers ofthe plurality of aptamers, wherein at least one aptamer of the pluralityof aptamers has less binding affinity for the first complex than atleast one of the second aptamers of the plurality of second complexes;e) dissociating the plurality of second complexes; f) quantifying theplurality of second aptamers to obtain a quantitative value; g)repeating steps a) through f) until the ratio of the quantitative valueto a reference value remains unchanged or decreases relative to theratio of the quantitative value to the reference value of the previousrepeating of steps a) through f), wherein the reference value is basedon quantifying a plurality of control aptamers exposed to the method ofsteps a) through f) without the target; h) identifying at least one ofthe aptamers of the plurality of second aptamers that is capable ofbinding the first complex.

In another aspect, the tag is biotin.

A. SELEX

SELEX generally includes preparing a candidate mixture of nucleic acids,binding of the candidate mixture to the desired target molecule to forman affinity complex, separating the affinity complexes from the unboundcandidate nucleic acids, separating and isolating the nucleic acid fromthe affinity complex, purifying the nucleic acid, and identifying aspecific aptamer sequence. The process may include multiple rounds tofurther refine the affinity of the selected aptamer. The process caninclude amplification steps at one or more points in the process. See,e.g., U.S. Pat. No. 5,475,096, entitled “Nucleic Acid Ligands”. TheSELEX process can be used to generate an aptamer that covalently bindsits target as well as an aptamer that non-covalently binds its target.See, e.g., U.S. Pat. No. 5,705,337 entitled “Systematic Evolution ofNucleic Acid Ligands by Exponential Enrichment: Chemi-SELEX.”

The SELEX process can be used to identify high-affinity aptamerscontaining modified nucleotides that confer improved characteristics onthe aptamer, such as, for example, improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX process-identified aptamers containing modifiednucleotides are described in U.S. Pat. No. 5,660,985, entitled “HighAffinity Nucleic Acid Ligands Containing Modified Nucleotides”, whichdescribes oligonucleotides containing nucleotide derivatives chemicallymodified at the 5′- and 2′-positions of pyrimidines. U.S. Pat. No.5,580,737, see supra, describes highly specific aptamers containing oneor more nucleotides modified with 2′-amino (2′-NH₂), 2′-fluoro (2′-F),and/or 2′-O-methyl (2′-OMe). See also, U.S. Pat. No. 8,409,795, entitled“SELEX and PHOTOSELEX”, which describes nucleic acid libraries havingexpanded physical and chemical properties and their use in SELEX andphotoSELEX.

SELEX can also be used to identify aptamers that have desirable off-ratecharacteristics. See, for example, U.S. Pat. No. 7,947,447, entitled“Method for Generating Aptamers with Improved Off-Rates”, whichdescribes improved SELEX methods for generating aptamers that can bindto target molecules. As mentioned above, these slow off-rate aptamersare known as “SOMAmers.” Methods for producing aptamers or SOMAmers andphotoaptamers or SOMAmers having slower rates of dissociation from theirrespective target molecules are described. The methods involvecontacting the candidate mixture with the target molecule, allowing theformation of nucleic acid-target complexes to occur, and performing aslow off-rate enrichment process wherein nucleic acid-target complexeswith fast dissociation rates will dissociate and not reform, whilecomplexes with slow dissociation rates will remain intact. Additionally,the methods include the use of modified nucleotides in the production ofcandidate nucleic acid mixtures to generate aptamers or SOMAmers withimproved off-rate performance.

A variation of this assay employs aptamers that include photoreactivefunctional groups that enable the aptamers to covalently bind or“photocrosslink” their target molecules. See, e.g., U.S. Pat. No.6,544,776 entitled “Nucleic Acid Ligand Diagnostic Biochip.” Thesephotoreactive aptamers are also referred to as photoaptamers. See, e.g.,U.S. Pat. No. 5,763,177, U.S. Pat. No. 6,001,577 and U.S. Pat. No.6,291,184, each of which is entitled “Systematic Evolution of NucleicAcid Ligands by Exponential Enrichment: Photoselection of Nucleic AcidLigands and Solution SELEX,” see also, e.g., U.S. Pat. No. 6,458,539,entitled “Photoselection of Nucleic Acid Ligands.” After the microarrayis contacted with the sample and the photoaptamers have had anopportunity to bind to their target molecules, the photoaptamers arephotoactivated, and the solid support is washed to remove anynon-specifically bound molecules. Harsh wash conditions may be used,since target molecules that are bound to the photoaptamers are generallynot removed, due to the covalent bonds created by the photoactivatedfunctional group(s) on the photoaptamers.

In both of these assay formats, the aptamers or SOMAmers are immobilizedon the solid support prior to being contacted with the sample. Undercertain circumstances, however, immobilization of the aptamers orSOMAmers prior to contact with the sample may not provide an optimalassay. For example, pre-immobilization of the aptamers or SOMAmers mayresult in inefficient mixing of the aptamers or SOMAmers with the targetmolecules on the surface of the solid support, perhaps leading tolengthy reaction times and, therefore, extended incubation periods topermit efficient binding of the aptamers or SOMAmers to their targetmolecules. Further, when photoaptamers or photoSOMAmers are employed inthe assay and depending upon the material utilized as a solid support,the solid support may tend to scatter or absorb the light used to effectthe formation of covalent bonds between the photoaptamers orphotoSOMAmers and their target molecules. Moreover, depending upon themethod employed, detection of target molecules bound to their aptamersor photoSOMAmers can be subject to imprecision, since the surface of thesolid support may also be exposed to and affected by any labeling agentsthat are used. Finally, immobilization of the aptamers or SOMAmers onthe solid support generally involves an aptamer or SOMAmer-preparationstep (i.e., the immobilization) prior to exposure of the aptamers orSOMAmers to the sample, and this preparation step may affect theactivity or functionality of the aptamers or SOMAmers.

SOMAmer assays that permit a SOMAmer to capture its target in solutionand then employ separation steps that are designed to remove specificcomponents of the SOMAmer-target mixture prior to detection have alsobeen described (see U.S. Pat. No. 7,855,054, entitled “MultiplexedAnalyses of Test Samples”). The described SOMAmer assay methods enablethe detection and quantification of a non-nucleic acid target (e.g., aprotein target) in a test sample by detecting and quantifying a nucleicacid (i.e., a SOMAmer). The described methods create a nucleic acidsurrogate (i.e., the SOMAmer) for detecting and quantifying anon-nucleic acid target, thus allowing the wide variety of nucleic acidtechnologies, including amplification, to be applied to a broader rangeof desired targets, including protein targets.

Embodiments of the SELEX process in which the target is a peptide aredescribed in U.S. Pat. No. 6,376,190, entitled “Modified SELEX ProcessesWithout Purified Protein.”

B. Slow Off-Rate Aptamers (SOMAmers)

Slow off-rate aptamers (SOMAmer reagents) have transformed the fields ofproteomics, biomarker discovery, and medical diagnostics. It is nowpossible to measure >1000 proteins simultaneously and with high accuracyin a small sample (0.1 mL) of serum, plasma, CSF, or tissue lysate. Theapplication of this highly multiplexed assay (SOMAscan) has led to thediscovery of biomarkers in infectious, pulmonary, oncological,cardiovascular, renal and neurological diseases. SOMAmers have expandedrange of protein targets and improved binding properties compared toconventional aptamers, because they contain deoxyuridine residues thatare modified at their 5-position with hydrophobic aromatic functionalgroups that mimic amino acid side-chains. SOMAmers are generated invitro by the SELEX process (Systematic Evolution of Ligands byExponential Enrichment) which consists of multiple rounds of selectionwith kinetic challenge, partitioning, and amplification.

C. Chemical Modifications to Aptamers

Aptamers may contain modified nucleotides that improve is properties andcharacteristics. Non-limiting examples of such improvements include, invivo stability, stability against degradation, binding affinity for itstarget, and/or improved delivery characteristics.

Examples of such modifications include chemical substitutions at theribose and/or phosphate and/or base positions of a nucleotide. SELEXprocess-identified aptamers containing modified nucleotides aredescribed in U.S. Pat. No. 5,660,985, entitled “High Affinity NucleicAcid Ligands Containing Modified Nucleotides,” which describesoligonucleotides containing nucleotide derivatives chemically modifiedat the 5′- and 2′-positions of pyrimidines. U.S. Pat. No. 5,580,737, seesupra, describes highly specific aptamers containing one or morenucleotides modified with 2′-amino (2′-NH₂), 2′-fluoro (2′-F), and/or2′-O-methyl (2′-OMe). See also, U.S. Pat. No. 8,409,795, entitled “SELEXand PHOTOSELEX,” which describes nucleic acid libraries having expandedphysical and chemical properties and their use in SELEX and photoSELEX.

Specific examples of a C-5 modification include substitution ofdeoxyuridine at the C-5 position with a substituent independentlyselected from: benzylcarboxyamide (alternatively benzylaminocarbonyl)(Bn), naphthylmethylcarboxyamide (alternativelynaphthylmethylaminocarbonyl) (Nap), tryptaminocarboxyamide(alternatively tryptaminocarbonyl) (Trp), and isobutylcarboxyamide(alternatively isobutylaminocarbonyl) (iBu) as illustrated immediatelybelow.

Chemical modifications of a C-5 modified pyrimidine can also be combinedwith, singly or in any combination, 2′-position sugar modifications,modifications at exocyclic amines, and substitution of 4-thiouridine andthe like.

Representative C-5 modified pyrimidines include:5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU),5-(N-benzylcarboxyamide)-2′-O-methyluridine,5-(N-benzylcarboxyamide)-2′-fluorouridine,5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),5-(N-isobutylcarboxyamide)-2′-O-methyluridine,5-(N-isobutylcarboxyamide)-2′-fluorouridine,5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-(N-tryptaminocarboxyamide)-2′-O-methyluridine,5-(N-tryptaminocarboxyamide)-2′-fluorouridine,5-(N-[1-(3-trimethylamonium)propyl] carboxyamide)-2′-deoxyuridinechloride, 5-[N-(1-naphthylmethyl)carboxyamide]-2′-deoxyuridine (NapdU),5-[N-(2-naphthylmethyl)carboxyamide]-2′-deoxyuridine (2-NapdU),5-(N-naphthylmethylcarboxyamide)-2′-O-methyluridine,5-(N-naphthylmethylcarboxyamide)-2′-fluorouridine or5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine.

If present, a modification to the nucleotide structure can be impartedbefore or after assembly of the polynucleotide. A sequence ofnucleotides can be interrupted by non-nucleotide components. Apolynucleotide can be further modified after polymerization, such as byconjugation with a labeling component.

Additional non-limiting examples of modified nucleotides (e.g., C-5modified pyrimidine) that may be incorporated into the nucleic acidsequences of the present disclosure include the following:

R′ is defined as follows:

And, R″, R′″ and R″″ are defined as follows:whereinR″″ is selected from the group consisting of a branched or linear loweralkyl (C1-C20); hydroxyl (OH), halogen (F, Cl, Br, I); nitrile (CN);boronic acid (BO₂H₂); carboxylic acid (COOH); carboxylic acid ester(COOR″); primary amide (CONH₂); secondary amide (CONHR″); tertiary amide(CONR″R′″); sulfonamide (SO₂NH₂); N-alkylsulfonamide (SONHR″);whereinR″, R′″ are independently selected from a group consisting of a branchedor linear lower alkyl (C1-C2)); phenyl (C₆H₅); an R″″ substituted phenylring (R″″C₆H₄); wherein R″″ is defined above; a carboxylic acid (COOH);a carboxylic acid ester (COOR′″″); wherein R′″″ is a branched or linearlower alkyl (C1-C20); and cycloalkyl; wherein R″═R′″═(CH₂)n;wherein n=2-10.

Further, C-5 modified pyrimidine nucleotides include the following:

wherein R is selected from one of the following moieties:

For nomenclature purposes and by way of example, where the R group isdefined as 6a (or Bn) above, the nucleotide is named5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU); where the R group isdefined as 6f (or Nap) above, the nucleotide is named5-[N-(1-naphthylmethyl)carboxyamide]-2′-deoxyuridine (NapdU) and wherethe R group is defined as 6 h (or 2Nap) above, the nucleotide is names5-[N-(2-naphthylmethyl)carboxyamide]-2′-deoxyuridine (2-NapdU).

In some embodiments, the modified nucleotide confers nuclease resistanceto the oligonucleotide. In some embodiments, the modified nucleotide isselected from the group consisting of chemical formulas 6a to 6v. Apyrimidine with a substitution at the C-5 position is an example of amodified nucleotide. Modifications can include backbone modifications,methylations, unusual base-pairing combinations such as the isobasesisocytidine and isoguanidine, and the like. Modifications can alsoinclude 3′ and 5′ modifications, such as capping. Other modificationscan include substitution of one or more of the naturally occurringnucleotides with an analog, internucleotide modifications such as, forexample, those with uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoamidates, carbamates, etc.) and those withcharged linkages (e.g., phosphorothioates, phosphorodithioates, etc.),those with intercalators (e.g., acridine, psoralen, etc.), thosecontaining chelators (e.g., metals, radioactive metals, boron, oxidativemetals, etc.), those containing alkylators, and those with modifiedlinkages (e.g., alpha anomeric nucleic acids, etc.). Further, any of thehydroxyl groups ordinarily present on the sugar of a nucleotide may bereplaced by a phosphonate group or a phosphate group; protected bystandard protecting groups; or activated to prepare additional linkagesto additional nucleotides or to a solid support. The 5′ and 3′ terminalOH groups can be phosphorylated or substituted with amines, organiccapping group moieties of from about 1 to about 20 carbon atoms,polyethylene glycol (PEG) polymers in one embodiment ranging from about10 to about 80 kDa, PEG polymers in another embodiment ranging fromabout 20 to about 60 kDa, or other hydrophilic or hydrophobic biologicalor synthetic polymers. In one embodiment, modifications are of the C-5position of pyrimidines. These modifications can be produced through anamide linkage directly at the C-5 position or by other types oflinkages.

Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclicsugar analogs, α-anomeric sugars, epimeric sugars such as arabinose,xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses,acyclic analogs and abasic nucleoside analogs such as methyl riboside.As noted above, one or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups includeembodiments wherein phosphate is replaced by P(O)S (“thioate”),

P(S)S (“dithioate”), (O)NR₂ (“amidate”), P(O)R, P(O)OR′, CO or CH₂(“formacetal”), in which each R or R′ is independently H or substitutedor unsubstituted alkyl (1-20 C) optionally containing an ether (—O—)linkage, aryl, alkenyl, cycloalky, cycloalkenyl or araldyl. Not alllinkages in a polynucleotide need be identical. Substitution ofanalogous forms of sugars, purines, and pyrimidines can be advantageousin designing a final product, as can alternative backbone structureslike a polyamide backbone, for example.D. Kits Comprising Compositions

The present disclosure provides kits comprising a first aptamer and/orsecond aptamer described herein. Such kits can comprise, for example,(1) a first aptamer (e.g., a target capture aptamer) and/or a secondaptamer (e.g., a target detection aptamer); and (2) at least onepharmaceutically acceptable carrier, such as a solvent or solution.Additional kit components can optionally include, for example: (1) anyof the pharmaceutically acceptable excipients identified herein, such asstabilizers, buffers, etc., (2) at least one container, vial or similarapparatus for holding and/or mixing the kit components; and (3) deliveryapparatus.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the disclosure to the particular features or embodimentsdescribed.

EXAMPLES Example 1: Materials and Methods

This example provides a summary of the general materials and methodsused to select and identify DNA aptamer pairs for target detection(e.g., protein target).

Proteins Used for SELEX.

C. difficile binary toxin (CdtA) was produced in recombinant,His₁₀-tagged form as described. Human proteins available in recombinant,tagged form (R&D Systems, Minneapolis, Minn., USA) includedangiopoietin-2 (Cat. No. 623-AN/CF), TSP2 (Cat. No. 1635-T2), CRDL1(Cat. No. 1808-NR), MATN2 (Cat. No. 3044-MN/CF), GPVI (Cat. No.3627-GP), which were His-tagged, and ESAM (Cat. No. 2688-EC) as anFc-fusion. Native human proteins purified from plasma included C7(Quidel, San Diego, Calif., USA, Cat. No. A405) and plasminogen (AthensResearch & Technology, Athens, Ga., USA, Cat. No. 16-16-161200); bothwere biotinylated using EZ-Link NHS-PEG4-Biotin (Thermo, Rockford, Ill.,USA, Cat. No 21329) as described.

Aptamer Synthesis.

Truncated synthetic aptamers that contained the 40-nucleotidetarget-binding region and five nucleotides on each end were prepared viastandard phosphoramidite chemistry using modified nucleotides. AB-H50mers contained a 5′-biotin-dA hexaethyleneglycol spacer for easycoupling to streptavidin (SA), and a 3′ inverted dT nucleotide (idT) forimproved exonuclease stability.

Menu Aptamers (SOMAmers) and Sandwich SELEX.

Menu aptamers as primary binding agents to all protein targets had beenisolated via SELEX and AB-H 50-mers were prepared as described above. Asan alternative to AB-H aptamers, PBDC (photocleavable linker with biotinand a flurophore) aptamers were used in a modified SELEX process. Theaptamers were “heat-cooled” to ensure their proper renaturation byheating to 95° C. for 3 minutes in SB18T and slowly cooling to 37° C.Activity was confirmed in equilibrium binding and in pull-down assays.For sandwich SELEX, the published selection protocol was modified asfollows. The proteins were complexed with the AB-H versions of thecognate menu aptamers immediately prior to each round of SELEX. For R1,100 μl of 500 nM protein (50 pmol) were mixed with 5 μl of 5 μM (25pmol) heat-cooled aptamers and incubated for 30 min at 37° C. to allowcomplex formation. For subsequent rounds, 10 μl of 500 nM protein (5pmol) and 2 μl of 5 μM (10 pmol) aptamers (2-fold excess) were used.Specific counter-selection beads to reduce background due tonon-specific aptamer-aptamer interactions were prepared fresh in eachround of SELEX. In brief, 2 μl of 5 μM (10 pmol) heat-cooled AB-Haptamers were added to 40 μl of 2.5 mg/ml SA beads in SB18T and shakenfor 15 minutes to allow immobilization. The beads were then washed toremove residual free aptamers, resuspended in 50 μl SB18T and added tothe counter-selection plate along with 10 μl Hexa-His (AnaSpec, Fremont,Calif., USA, Cat. No 24420) coated beads, or with 50 μl SA beads orProtein G beads according to the downstream partitioning method. BufferSB18T (40 mM HEPES pH 7.5, 0.1 M NaCl, 5 mM KCl, 5 mM MgCl₂, 0.05%Tween-20) was used for SELEX and for all subsequent binding assays. Thestarting library consisted of 1 nmol (10¹⁴-10¹⁵) sequences of modifiedDNA sequences containing 40 consecutive randomized positions flanked byfixed sequences for PCR amplification. Separate libraries with differentmodified nucleotides were used, including5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-(N-[2-naphthylmethyl]carboxyamide)-2′-deoxyuridine (2NapdU), and5-(N-phenethylcarboxyamide)-2′-deoxyuridine (PEdU). A kinetic challengewith 5 mM dextran sulfate was performed from SELEX round 2 forward tofavor slow off-rates. Partitioning of the target-aptamer complexes wasachieved with paramagnetic Dynabeads® (Life Technologies, Carlsbad,Calif., USA), using His-tag 2 (Cat. No. 101-04D) for His-taggedproteins, Protein G (Cat. No. 100-04D) for Fc-fusion proteins), andMyOne Streptavidin C1 (Cat. No. 350-02D) beads for biotinylated targets,respectively. Selected DNA was eluted from the beads with sodiumperchlorate elution buffer (1.8 M NaClO₄, 40 mM PIPES, 1 mM EDTA, 0.05%Triton X-100, pH 6.8) for 5 min, then captured on primer beads andprocessed for PCR and primer extension to obtain the sense-strands withthe modified nucleotides using KOD XL DNA polymerase.

For the modified sandwich SELEX, partitioning of the target-aptamercomplex was accomplished with streptavidin (SA) beads that bind thebiotin-tag on the primary PBDC aptamer. This is in contrast to the AB-Haptamer based SELEX, where the biotin-tag is on the target. Thismodified method allowed for the use of untagged targets in sandwichSELEX. The selected DNA aptamers from the library were harvested viaphotocleavage (2 times at 7 minutes with shaking under a blacklight) oftripartite complexes from the SA beads instead of elution with sodiumperchlorate, and processed for PCR and eDNA preparation.

DNA Sequencing and Comparative Sequence Analysis.

Aptamer pools obtained in SELEX were cloned using the PCR-Script AmpCloning Kit (Agilent, Santa Clara, Calif., USA, Cat. No. 211189) andsequences of individual clones were determined on an ABI Prism 3730(SeqWright, Houston, Tex.). Aptamers obtained in SELEX with complexed vsfree protein were compared to identify common patterns, usingcustomized, flexible alignment algorithms with pattern identitythreshold=0.5-0.9, family cluster cutoff=0.5-0.9, sequence matchthreshold=0.8, and equivalence mismatches=5.

Equilibrium Binding Assays and Competition Assays.

Full-length aptamers isolated in SELEX and their synthetic, truncatedcounterparts were first evaluated for binding to free protein and thento pre-formed protein-menu aptamer complexes (stoichiometric ratio 1:1)to determine and compare the equilibrium binding constants, K_(d)'s, infilter-binding assays. Efficient partitioning of the complexes ontonylon membranes was achieved with Zorbax PSM-300A (Agilent, Santa Clara,Calif., USA), except for complexes with GPVI, where Dynabeads® His-tag 2(Life Technologies, Carlsbad, Calif., USA, Cat. No. 101-04D) were usedinstead. Sandwich aptamer candidates were further tested in competitionbinding assays, where radiolabeled aptamers (˜0.01 nM) were incubatedwith target proteins over a range of concentrations (0.001 nM to 100 nM)in the presence of 100-fold excess (10 nM) unlabeled menu aptamer ascompetitors.

Sandwich Filter-Binding Assays.

Two variations of filter-binding sandwich assays were performed toobtain 12-pt binding curves. The first method involved equilibriumbinding of protein and radiolabeled, full-length detection aptamer,followed by partitioning (30 min with intermittent shaking) withspecific capture beads that were prepared by immobilizing AB-H menuaptamers on SA beads. The second method was based on the formation oftripartite complexes during equilibrium binding of protein, radiolabeleddetection aptamer (full-length, non-biotinylated) and excess (10 nM)unlabeled, AB-H menu aptamer, followed by partitioning (5 min withintermittent shaking) with SA beads. Both methods yielded comparableresults. As controls, all sequences were tested in separate assays wherethe capture agent was omitted, to identify non-specific background dueto SA bead binding. These clones, along with sequences resulting innon-titratable signals due to direct interaction of capture anddetection aptamer, were removed from further analysis.

Multiplexed Sandwich Screening Assays.

The Luminex platform was used for the multiplexed, pair-wise screeningof aptamers (AB-H 50mers) in sandwich binding format. Capture beadpreparation and binding assays were performed in MSBVN12 filter plates(EMD Millipore Corp., Billerica, Mass., USA) pre-wet with buffer SB17T(SB18T supplemented with 1 mM EDTA). Different types of LumAvidin®Microspheres (Luminex Corporation, Austin, Tex., Cat. No. L100-L101-01through L100-L116-01) were dispensed into separate wells (100,000 beadsper well) and washed 3×1 min with 180 μl SB17T by vacuum filtration.Aptamers were heat-cooled, 80 μl of 50 nM stocks of each capture aptamerfor a given protein target were added to a different bead type, and theplate was shaken (20 min at RT, 1100 rpm) to allow for immobilization.The beads were then washed for 5 min each with 100 μl 50 nM streptavidinand with 10 mM biotin in SB17T, then 4×1 min with 180 μl SB17T. Allcapture beads for each protein were pooled and the volume brought to 1.7ml with SB17T. For the binding assay, 50 μl of pooled capture beads weredispensed into the wells of a pre-wet MSBVN12 filter plate, usingduplicate wells for each aptamer to be tested as detection agent, andmixed with 50 μl of 20 nM protein in SB17T+1% BSA or 50 μl buffer forthe no-protein controls. After at least 30 min with shaking at 1100 rpm,the plate was vacuum-washed 2×1 min with 180 μl SB17T+1% BSA and thebeads were resuspended in 50 μl SB17T+BSA. Heat-cooled detectionaptamers were added to individual wells, using 50 μl of 12.5 nM stocks,and incubation was continued for 30 min with shaking, then the beadswere washed and resuspended as above. As a reporter, 50 μl of 10 μg/mlstreptavidin-phycoerythrin (SA-PE) conjugate (Moss, Pasadena, Md., USA,Cat. No. SAPE-001) in SB17T+BSA were added, and the beads were againshaken, washed, and resuspended as above. The plate was read on aLuminex 100 analyzer (time out: enabled 120 s, DD gating: 7500-8000,reporter gain: high PMT).

Sandwich Assay Target Titrations.

Binding curves for AB-H aptamer pairs were generated for both abead-based and plate-based sandwich assay. For the bead-assay, a singleLumAvidin microsphere bead type carrying one specific capture aptamerwas used, and the assay was performed essentially as described for thescreening assay above, except that target protein was added in half-logserial dilutions starting at 100 nM to obtain 12-point binding curves.For the plate-assay, 2 pmol (100 μl of 20 nM) heat-cooled aptamers wereimmobilized overnight on Reacti-Bind Streptavidin Coated Plates (PierceBiotechnology-Thermo Scientific, Rockford, Ill., USA, Cat. No. 15500).The wells were washed for 5 min each with 100 μl 50 nM streptavidin andwith 10 mM biotin in SB17T, then blocked with 200 μl SB17T+1% BSA for 10min. Target proteins were added and incubated for 45 min with shaking,and the plate was washed 2×1 min with 150 μl SB17T+1% BSA. Detectionaptamers were added (100 μl of 20 nM stocks in SB17T+1% BSA), incubationwas continued for 35 minutes, and the wells washed as above. Asreporter, 100 μl of 0.4 μg/ml SA-HRP conjugate (Life Technologies,Carlsbad, Calif., USA, Cat. No. S-911) was added for 35 minutes withshaking, followed by three washes with SB17 (no Tween-20). TMB substrate(FisherScientific, Pittsburgh, Pa., USA, Cat. No. PI34028) was added(100 μl) for 20-30 min, then the reaction was stopped with 50 μl of 10%sulfuric acid, and absorbance at 450 nm was recorded.

Example 2: Selection and Identification of Aptamer Pairs for BinaryToxin A Chain (CdtA) of Clostridium Difficile

This example provides the representative method for the selection andproduction of DNA aptamer pairs for the binary toxin A chain (CdtA)protein of C. difficile. This representative method is outlined in FIG.1 and may be used to identify aptamer pairs for other targets (e.g.,protein) of interest.

SELEX with purified recombinant CdtA protein and a TrpdU-modifiedlibrary yielded clone 4758-6 having a K_(d) of 0.86 nM. The nucleic acidmolecule of clone 4758-6 is as an aptamer forty (40) nucleotides inlength comprising C-5 modified pyrimidines, specifically TrpdU, and iscapable of binding to the CdtA protein. The nucleotide sequence is asfollows: 5′-

(SEQ ID NO: 1) GAAGACTTTAATTCTGACATGGTGTCCAATGGCGCGCGAG-3′,with T represents a TrpdU. In an attempt to identify a non-competingaptamer, CdtA in a complex with a non-amplifiable version of clone4758-6 (CdtA-4758-6 aptamer complex) was used as the target in a secondSELEX (pool 5579-2NapdU modified aptamer library), which employed a2NapdU-modified library instead of the TrpdU library used to generatethe 4758-6 clone. Table 1 below provides a summary of the sequenceanalysis of the aptamers (or clones) identified in SELEX using the4758-6 aptamer clone complexed with the CdtA protein, the correspondingnumber of sequence patterns identified (“#”), K_(d) data and whether aaptamer-sandwich formed with the CdtA-4758-6 aptamer complex(“Sandwich”). Abbreviations: free protein (F.P.) and competitor (Comp.).

TABLE 1 CdtA-4758-6 complex SELEX pool 5579 (2NapdU mod.) (n = 45evaluable sequences) K_(d) (nM) K_(d) (nM) # Clone ID F.P. w/Comp.Sandwich 20 5579-12 0.97 0.28 Yes 4 5579-5 2.24 0.21 Yes 12 5579-11 0.070.71 No 5 5579-8 0.53 0.55 Yes 2 5579-10 0.15 0.13 Yes 2 NT² NT NT NT²NT, not tested *eDNA full-length SOMAmer testing data (no syntheticSOMAmers produced)

SELEX with free CdtA (pool 5551) was done in parallel with the same2NapdU library used in SELEX against the CdtA-4758-6 aptamer complex,and also with a different PEdU modified library (pool 5574). Followingeight rounds of selection, all SELEX experiments were successful ingenerating sequence pools with at least 100-fold affinity enrichmentcompared to starting random libraries. Table 2 below provides a summaryof the sequence analysis of the aptamers (or clones) identified in SELEXusing the free CdtA protein (pool 5551-2NapdU modified aptamer library),the corresponding number of sequence patterns identified (“#”), K_(d)data and whether a aptamer-sandwich formed (“Sandwich”).

TABLE 2 Free CdtA SELEX pool 5551 (2NapdU mod.) (n = 43 evaluablesequences) K_(d) (nM) K_(d) (nM) # Clone ID F.P. w/Comp. Sandwich 65551-81 0.54 0.55 Yes 4 5551-50 0.09 17.60 No 15 5551-52 0.14 0.22 No 85551-51 Bead binder NT NT 5 5551-49 0.31 20.60 No 2 5551-76 NT NT NT 25551-82 NT NT NT 1 NT NT NT NT ²NT, not tested *eDNA full-length SOMAmertesting data (no synthetic SOMAmers produced)

Table 3 below provides a summary of the sequence analysis of theaptamers (or clones) identified in SELEX using the free CdtA protein(pool 5574—PEdU modified aptamer library), the corresponding number ofsequence patterns identified (“#”), K_(d) data and whether aaptamer-sandwich formed (“Sandwich”).

TABLE 3 Free CdtA SELEX pool 5574 (PEdU mod) (n = 41 evaluablesequences) K_(d) (nM) K_(d) (nM) # Clone ID F.P. w/Comp. Sandwich 135574-49 1.5  >10 No 11 5574-56 2.92* >10 No 10 5574-51 >10*    >10 NT 25574-67 4.20* >10 NT 2 5574-83 4.66* >10 NT 3 NT NT NT NT ²NT, nottested *eDNA full-length SOMAmer testing data (no synthetic SOMAmersproduced)

Comparative sequence analysis of the 2NapdU clones fromaffinity-enriched pools 5579 (CdtA-4758-6; Table 1) and 5551 (free CdtA;Table 2) revealed differences in the patterns and abundance, but therewere some shared sequence motifs as well (FIG. 1B). The dominant patternoccurred in 20 (44%) of the sequences in pool 5579, but only in 5 (12%)of the sequences in pool 5551. Sequences harboring this pattern (e.g.,5579-7, 5579-12, 5579-21) performed consistently well in sandwich formatwith the original ligand 4758-6. Another pattern and two sequences foundin multiple copies, were found exclusively in pool 5579 (CdtA-4758-6),and also these sequences were non-competing with 4758-6. Another patternwas present in sequences from both pools, and this family contained themost active aptamers (e.g. 5579-11), but they failed in the sandwichassay. Most likely, these sequences successfully competed with 4758-6during SELEX, occupying the same epitope with higher affinity orsuperior binding kinetics.

Two different patterns and three multicopy sequences were found only inpool 5551 (free CdtA SELEX) and failed the sandwich assay. Finally, noneof the sequences generated in free CdtA SELEX using a PEdU library boundthe complex, although several of them had sub-nanomolar affinity to freeCdtA. Thus, SELEX with the CdtA-4758-6 complex clearly resulted in ahigher fraction of sequences useful for sandwich assays in conjunctionwith 4758-6, although free CdtA SELEX also produced a few new aptamersthat bound to a different epitope.

The new aptamers bound the free protein with affinities ranging from0.05 nM-14 nM (FIG. 1C), and were also tested in competition assays andin sandwich format together with the existing 4758-6 TrpdU aptamer(FIGS. 1D and 1E). As shown for clone 5579-12, which possessed affinitycomparable to 4758-6 (K_(d)=0.67 nM vs 0.86 nM), binding to CdtA was notaffected by the presence of a ˜100-fold excess (10 nM) 4758-6competitor, or when 4758-6 was used as a capture agent, indicating thatthe two sequences bind to distinct CdtA epitopes. In contrast, bindingof aptamer 5579-11, which had superior affinity compared to 4758-6(K_(d)=0.05 nM vs 0.86 nM), was reduced with 4758-6 as competitor orcapture agent. Representative sequences from each SELEX were alsoscreened on the Luminex platform, where the first aptamer 4758-6 servedas capture agent on beads. Most clones from complex SELEX (5579-7,5579-10, 5579-12, 5579-21) as well as one clone from free CdtA SELEX(5551-81) confirmed their binding when used as detection agent (FIG.1F). Capture and detection agents were interchangeable for 4758-6 and5579-12, producing similar binding curves in the Luminex sandwich assay(FIG. 1G).

Example 3: Selection of Aptamer Pairs for Eight Different ProteinTargets

This example provides the representative method for the selection andproduction of DNA aptamer pairs for the following protein targets:angiopoietin-2 (ANGPT2), thrombospondin-2 (TSP2), chordin-like 1(CRDL1), matrilin-2 (MATN2), glycoprotein VI (GPVI), endothelialcell-selective adhesion molecule (ESAM), complement 7 (C7), andplasminogen (PLG).

A two-tiered strategy was employed to obtain aptamer pairs to the targetproteins. For the first strategy, aptamers obtained via SELEX thatdemonstrated good affinity (K_(d)<10 nM) yielded functional aptamerpairs for three of the eight targets (C7, MATN2, PLG), all with BndU asthe modified nucleotide. For the second strategy, SELEX was performedwith target-SOMAmer complexes, employing two new modified nucleotidelibraries (TrpdU and 2NapdU), and, in parallel a free protein SELEXusing either a TrpdU or 2NapdU library. After seven rounds of SELEX, all24 pools showed convergence based on DNA reassociation (C₀t) kineticsand demonstrated low-nanomolar or sub-nanomolar K_(d) values. Cloningand routine sequencing of at least 48 SOMAmers per pool allowedcomparative analysis of sequences obtained in SELEX with protein-SOMAmercomplex and free protein targets. In all cases, active clones that boundthe protein-SOMAmer complexes were obtained, however, a larger fractionof the tested clones did not bind the complex where the free target wasused for SELEX. Therefore, using protein-SOMAmer complex targets duringSELEX clearly increased the likelihood of finding sandwich candidates.An exception, however, were clones from pool 7565 TrpdU selected withthe ESAM-2981-9 complex, all of which shared a common sequence patternand showed binding of the ESAM-2981-9 complex but not free ESAM protein.These sequences were later shown to interact with SOMAmer 2981-9 ratherthan bind the target protein.

The observed binding of the new clones to the protein-menu SOMAmercomplexes did not prove the existence of a true sandwich, since theymight simply displace the menu SOMAmer and bind to the same epitope. Tomake this distinction, the new clones were subjected to binding assaysin the presence of 10 nM (˜100-fold) excess unlabeled competitor menuSOMAmer and in sandwich assays with the menu SOMAmers as capture agents.The sandwich assay depicted in FIG. 2A results in a signal only if asandwich is formed, but not if displacement of the first SOMAmer occurs.Detailed sequence analysis and binding characteristics for all SOMAmers(synthetic 5′AB-H 50mers) obtained in the SELEX with complexed or freeproteins are shown below in Tables 4-27.

Tables 4-6 provide a summary for the aptamers to the angiopoietin-2(ANGPT2) protein. The nucleic acid molecule of clone 2602-2 is as anaptamer forty (40) nucleotides in length comprising C-5 modifiedpyrimidines, specifically BndU, and is capable of binding to the ANGPT2protein.

TABLE 4 ANGPT2-2602-2 complex SELEX pool 7560 (TrpdU mod.) (n = 43evaluable sequences) K_(d) (nM) K_(d) (nM) # Clone ID F.P. w/Comp.Sandwich 26 7560-4 0.29 0.59 Yes 4 7560-1 0.19 1.26 Yes 4 7560-19 6.352.65 Yes 2 7560-27 >100*    NT NT 7 NT NT NT NT ²NT, not tested *eDNAfull-length SOMAmer testing data (no synthetic SOMAmers produced)

TABLE 5 Free ANGPT2 SELEX pool 7568 (TrpdU mod.) (n = 80 evaluablesequences) K_(d) (nM) K_(d) (nM) # Clone ID F.P. w/Comp. Sandwich 297568-4  0.88* NT NT 4 7568-87 NT NT NT 15 7568-30 0.15 0.15 Yes 47568-29 0.50 1.05 Yes 4 7568-53 0.78 0.65 Yes 2 7568-1 3.14 5.24 Yes 27568-12 >100*    NT NT 2 7568-14  6.84* NT NT 2 7568-15 NT NT NT 27568-25 NT NT NT 14 NT NT NT NT ²NT, not tested *eDNA full-lengthSOMAmer testing data (no synthetic SOMAmers produced)

TABLE 6 ANGPT2-2602-2 complex SELEX pool 7573 (2NapdU mod.) (n = 42evaluable sequences) K_(d) (nM) K_(d) (nM) # Clone ID F.P. w/Comp.Sandwich 9 7573-14 0.21 0.22 Yes 4 7573-6 >10 NT NT 2 7573-15 0.82 0.98Yes 8 7573-1 0.12 0.17 Yes 3 7573-42 0.07 0.09 Yes 3 7573-21 0.16 0.54Yes 3 7573-22 1.5 NT NT 2 7573-12 NT NT NT 8 NT NT NT NT ²NT, not tested*eDNA full-length SOMAmer testing data (no synthetic SOMAmers produced)

Tables 7-9 provide a summary for the aptamers to the thrombospondin-2(TSP2) protein. The nucleic acid molecule of clone 3339-33 is as anaptamer forty (40) nucleotides in length comprising C-5 modifiedpyrimidines, specifically BndU, and is capable of binding to the TSP2protein.

TABLE 7 TSP2-3339-33 complex SELEX pool 7561 (TrpdU mod.) (n = 41evaluable sequences) K_(d) (nM) K_(d) (nM) # Clone ID F.P. w/Comp.Sandwich 8 7561-59 0.19 0.14 Yes 5 7561-69 NT NT NT 9 7561-55 0.04 0.04Yes 3 7561-49 NT NT NT 2 7561-83 0.02 0.05 Yes 7 7561-65 0.09 3.08 No 7NT NT NT NT ²NT, not tested *eDNA full-length SOMAmer testing data (nosynthetic SOMAmers produced)

TABLE 8 Free TSP2 SELEX pool 7569 (TrpdU mod.) (n = 43 evaluablesequences) K_(d) (nM) K_(d) (nM) # Clone ID F.P. w/Comp. Sandwich 37569-1 NT NT NT 5 7569-23 NT NT NT 5 7569-29 0.03 0.04 Yes 5 7569-220.43 3.10 Yes 5 7569-18  0.05*  0.13* NT 3 7569-33 NT NT NT 3 7569-45 NTNT NT 2 7569-6 0.19 0.25 NT 2 7569-16 NT NT NT 10 NT NT NT NT ²NT, nottested *eDNA full-length SOMAmer testing data (no synthetic SOMAmersproduced)

TABLE 9 TSP2-3339-33 complex SELEX pool 7574 (2NapdU mod.) (n = 42evaluable sequences) K_(d) (nM) K_(d) (nM) # Clone ID F.P. w/Comp.Sandwich 11 7574-62 0.09 0.08 Yes 20 7574-57 0.03 0.08 Yes 5 7574-530.08 0.07 Yes 3 7574-64 0.18 1.10 Yes 3 NT NT NT NT ²NT, not tested*eDNA full-length SOMAmer testing data (no synthetic SOMAmers produced)

Tables 10-12 provide a summary for the aptamers to the chordin-like 1(CRDL1) protein. The nucleic acid molecule of clone 3362-61 is as anaptamer forty (40) nucleotides in length comprising C-5 modifiedpyrimidines, specifically BndU, and is capable of binding to the CRDL1protein.

TABLE 10 CRDL1-3362-61 complex SELEX pool 7562 (TrpdU mod.) (n = 47evaluable sequences) K_(d) (nM) K_(d) (nM) # Clone ID F.P. w/Comp.Sandwich 4 7562-3 0.13 0.23 Yes 4 7562-24 0.34 0.23 Yes 4 7562-8 0.380.31 Yes 5 7562-4 NT NT NT 5 7562-23 0.68 0.38 Yes 2 7562-6 0.44 0.17Yes 3 7562-12 0.12 0.49 Yes 3 7562-31 0.26 0.17 Yes 2 7562-2 0.28 0.13Yes 2 7562-7 1.52 0.23 Yes 2 7562-19 0.29 0.27 Yes 11 NT NT NT NT ²NT,not tested *eDNA full-length SOMAmer testing data (no synthetic SOMAmersproduced)

TABLE 11 Free CRDL1 SELEX pool 7570 (TrpdU mod.) (n = 41 evaluablesequences) K_(d) (nM) K_(d) (nM) # Clone ID F.P. w/Comp. Sandwich 27570-90 NT NT NT 1 7570-64 NT NT NT 4 7570-67 0.30* 0.28* NT 6 7570-500.50* 2.44* NT 5 7570-52 0.89* 2.14* NT 2 7570-57 0.74* 3.13* NT 47570-55 0.40  0.13  Yes 2 7570-53 0.41* 1.07* NT 2 7570-84 NT NT NT 13NT NT NT NT ²NT, not tested *eDNA full-length SOMAmer testing data (nosynthetic SOMAmers produced)

TABLE 12 CRDL1-3362-61 complex SELEX pool 7575 (2NapdU mod.) (n = 42evaluable sequences) K_(d) (nM) K_(d) (nM) # Clone ID F.P. w/Comp.Sandwich 9 7575-2 0.38 0.19 Yes 7 7575-6 0.41 0.43 Yes 5 7575-19 3.510.13 Yes 2 7575-5 0.30 0.19 Yes 3 7575-3 0.55 3.36 NT 16 NT NT NT NT²NT, not tested *eDNA full-length SOMAmer testing data (no syntheticSOMAmers produced)

Tables 13-15 provide a summary for the aptamers to the matrilin-2(MATN2) protein. The nucleic acid molecule of clone 3325-2 is as anaptamer forty (40) nucleotides in length comprising C-5 modifiedpyrimidines, specifically BndU, and is capable of binding to the MATN2protein.

TABLE 13 MATN2-3325-2 complex SELEX pool 7563 (TrpdU mod.) (n = 40evaluable sequences) K_(d) (nM) K_(d) (nM) # Clone ID F.P. w/Comp.Sandwich 4 7563-61 NT NT NT 10 7563-63  0.17* 13.6* NT 8 7563-60 12.200.78 Yes 3 7563-51  3.11 1.19 NT 3 7563-55 NT NT NT 2 7563-56 >10*  1.75* NT 2 7563-58 NT NT NT 8 NT NT NT NT ²NT, not tested *eDNAfull-length SOMAmer testing data (no synthetic SOMAmers produced)

TABLE 14 Free MATN2 SELEX pool 7571 (TrpdU mod.) (n = 46 evaluablesequences) K_(d) (nM) K_(d) (nM) # Clone ID F.P. w/Comp. Sandwich 57571-11 0.66* 15.4*  NT 2 7571-31 5.31 3.65 Yes 4 7571-20 1.18 0.96 Yes4 7571-1 0.33* >10*    NT 4 7571-12 3.99* >10*    NT 27 NT NT NT NT ²NT,not tested *eDNA full-length SOMAmer testing data (no synthetic SOMAmersproduced)

TABLE 15 MATN2-3352-2 complex SELEX pool 7576 (2NapdU mod.) (n = 90evaluable sequences) K_(d) (nM) K_(d) (nM) # Clone ID F.P. w/Comp.Sandwich 31 7576-61 0.63 0.49 Yes 10 7576-40 2.70 1.70 No 3 7576-130.94* 1.40* NT 11 7576-11 >100 6.53 Yes 5 7576-64 1.33 0.83 No 4 7576-194.57 2.08 Yes 4 7576-30 5.00 3.79 Yes 2 7576-51 0.30 0.46 Yes 20 NT NTNT NT ²NT, not tested *eDNA full-length SOMAmer testing data (nosynthetic SOMAmers produced)

Tables 16-18 provide a summary for the aptamers to the glycoprotein VI(GPVI) protein. The nucleic acid molecule of clone 3194-36 is as anaptamer forty (40) nucleotides in length comprising C-5 modifiedpyrimidines, specifically BndU, and is capable of binding to the GPVIprotein.

TABLE 16 GPVI-3194-36 complex SELEX pool 7564 (TrpdU mod.) (n = 82evaluable sequences) K_(d) (nM) K_(d) (nM) # Clone ID F.P. w/Comp.Sandwich 24 7564-5  0.04 0.02 Yes 20 7564-29  0.02*  0.35* Yes 8 7564-180.08 0.12 No 8 7564-3  0.07 0.18 No 2  7564-153 NT NT NT 9  7564-1740.04 0.42 NT 5 7564-13 NT NT NT 6 NT NT NT NT ²NT, not tested *eDNAfull-length SOMAmer testing data (no synthetic SOMAmers produced)

TABLE 17 Free GPVI SELEX pool 7572 (TrpdU mod.) (n = 44 evaluablesequences) K_(d) (nM) K_(d) (nM) # Clone ID F.P. w/Comp. Sandwich 67572-68 NT NT NT 6 7572-61 NT NT NT 2 7572-70 NT NT NT 7 7572-600.12 >10 NT 4 7572-79 0.03 1.83 NT 2 7572-49 NT NT NT 17 NT NT NT NT²NT, not tested *eDNA full-length SOMAmer testing data (no syntheticSOMAmers produced)

TABLE 18 GPVI-3194-36 complex SELEX pool 7577 (2NapdU mod.) (n = 44evaluable sequences) K_(d) (nM) K_(d) (nM) # Clone ID F.P. w/Comp.Sandwich 4 7577-51 >10 0.10 Yes 7 7577-70 >10 1.32 NT 4 7577-49 >3213.3  NT 6 7577-65 NT NT NT 3 7577-50 0.04 0.92 No 20 NT NT NT NT ²NT,not tested *eDNA full-length SOMAmer testing data (no synthetic SOMAmersproduced)

Tables 19-21 provide a summary for the aptamers to the endothelialcell-selective adhesion molecule (ESAM) protein. The nucleic acidmolecule of clone 2981-9 is as an aptamer forty (40) nucleotides inlength comprising C-5 modified pyrimidines, specifically BndU, and iscapable of binding to the ESAM protein.

TABLE 19 ESAM-2981-9 complex SELEX pool 7565 (T = TrpdU) (n = 44evaluable sequences) K_(d) (nM) K_(d) (nM) # Clone ID F.P. w/Comp.Sandwich 33 7565-67 >10 0.53 No 7 7565-54 >10 4.40 No 4 NT NT NT NT ²NT,not tested *eDNA full-length SOMAmer testing data (no synthetic SOMAmersproduced)

TABLE 20 ESAM-2981-9 complex SELEX pool 7578 (T = 2NapdU) (n = 42evaluable sequences) K_(d) (nM) K_(d) (nM) # Clone ID F.P. w/Comp.Sandwich 9 7578-5  0.18 0.43 No 8 7578-34 >10 0.56 No 8 7578-22 >10 1.57NT 3 7578-3  0.37 15.3 NT 2 7578-11 0.45 4.54 NT 12 NT NT NT NT ²NT, nottested *eDNA full-length SOMAmer testing data (no synthetic SOMAmersproduced)

TABLE 21 Free ESAM SELEX pool 7581 (T = 2NapdU) (n = 85 evaluablesequences) K_(d) (nM) K_(d) (nM) # Clone ID F.P w/Comp. Sandwich 17581-2 NT NT NT 3  7581-42 NT NT NT 12  7581-41 0.03 0.06 No 9 7581-50.48 3.58 NT 13 7581-8 0.44 3.55 NT 10 7581-3 0.40 2.32 NT 7  7581-540.24 3.00 NT 4 7581-9 0.16 1.99 NT 26 NT NT NT NT ²NT, not tested *eDNAfull-length SOMAmer testing data (no synthetic SOMAmers produced)

Tables 22-24 provide a summary for the aptamers to the complement 7 (C7)protein. The nucleic acid molecule of clone 2888-49 is as an aptamerforty (40) nucleotides in length comprising C-5 modified pyrimidines,specifically BndU, and is capable of binding to the C7 protein.

TABLE 22 C7-2888-49 complex SELEX pool 7566 (TrpdU mod.) (n = 46evaluable sequences) K_(d) (nM) K_(d) (nM) # Clone ID F.P w/Comp.Sandwich 6 7566-14 47.0 5.04 Yes 4 7566-40 0.14*  0.05* No 187566-22 >10 Bead binder NT 2 7566-29 13.3 10.4  NT 16 NT NT NT NT ²NT,not tested *eDNA full-length SOMAmer testing data (no synthetic SOMAmersproduced)

TABLE 23 C7-2888-49 complex SELEX pool 7579 (2NapdU mod.) (n = 43evaluable sequences) K_(d) (nM) K_(d) (nM) # Clone ID F.P w/Comp.Sandwich 2 7579-65 0.76* 0.04* Yes 2 7579-67 0.40* 0.09* Yes 9 7579-82NT NT NT 1 7579-68 >10 >10 NT 8 7579-88 0.19 0.17 No 3 7579-64 0.22 0.13No 2 7579-85 13.2 13.8 NT 16 NT NT NT NT ²NT, not tested *eDNAfull-length SOMAmer testing data (no synthetic SOMAmers produced)

TABLE 24 Free C7 SELEX pool 7582 (2NapdU mod.) (n = 42 evaluablesequences) K_(d) (nM) K_(d) (nM) # Clone ID F.P w/Comp. Sandwich 27582-52 NT NT NT 22 7582-56 >10 >10 NT 2 NT NT NT NT 5 7582-67 NT NT NT2 7582-52 NT NT NT 9 NT NT NT NT ²NT, not tested *eDNA full-lengthSOMAmer testing data (no synthetic SOMAmers produced)

Tables 25-27 provide a summary for the aptamers to the plasminogen (PLG)protein. The nucleic acid molecule of clone 4151-6 is as an aptamerforty (40) nucleotides in length comprising C-5 modified pyrimidines,specifically BndU, and is capable of binding to the PLG protein.

TABLE 25 PLG-4151-6 complex SELEX pool 7567 (TrpdU mod.) (n = 42evaluable sequences) K_(d) (nM) K_(d) (nM) # Clone ID F.P w/Comp.Sandwich 12 7567-53 >10 Bead binder NT 5 7567-63 2.41 1.95 NT 37567-95 >10 Bead binder NT 2 7567-64 0.99 0.30 Yes 20 NT NT NT NT ²NT,not tested *eDNA full-length SOMAmer testing data (no synthetic SOMAmersproduced)

TABLE 26 PLG-4151-6 complex SELEX pool 7580 (2NapdU mod.) (n = 42evaluable sequences) K_(d) (nM) K_(d) (nM) # Clone ID F.P w/Comp.Sandwich 1 7580-16 NT NT NT 2 7580-35 NT NT NT 1 7580-17 NT NT NT 27580-19 0.77 1.14 Yes 2 7580-13 1.75 1.37 Yes 11 7580-4  >10 Bead binderNT 7 7580-43 1.89 1.51 NT 16 NT NT NT NT ²NT, not tested *eDNAfull-length SOMAmer testing data (no synthetic SOMAmers produced)

TABLE 27 Free PLG SELEX pool 7583 (2NapdU mod.) (n = 43 evaluablesequences) K_(d) (nM) K_(d) (nM) # Clone ID F.P w/Comp. Sandwich 37583-19 3.72 0.72 Yes 8 7583-8  1.95* 0.86* NT 3 7583-7  3.91* 2.30* NT2 7583-15 1.14 0.21 Yes 2 7583-3  0.81 0.30 Yes 2 7583-24 8.72 0.59 Yes2 7583-12 1.94* 1.69* NT 21 NT NT NT NT ²NT, not tested *eDNAfull-length SOMAmer testing data (no synthetic SOMAmers produced)

While this binding assay was useful to identify aptamers that perform insandwich format, it is limited to pair-wise testing. Thus, we set up ahighly multiplexed sandwich screening assay method using the Luminexplatform (FIG. 2B), as described in detail in Example 1.

Example 4: Selection of Aptamer Pairs for Target Proteins by a ModifiedSandwich SELEX Method

This example provides the representative method for the selection andproduction of DNA aptamer pairs for the following protein targets:matrix metalloproteinase-12 (MMP-12) and secreted phospholipase A2(NPS-PLA2).

Aptamer pairs to the target proteins were obtained following a modifiedsandwich SELEX protocol that employed PBDC aptamers as capture agentsattached on beads for partitioning of target-aptamer complexes, followedby photocleavage of the tripartite complexes. For MMP-12, the PBDCaptamer 4496-60 BndU was used to form a complex with the target forSELEX using a NapdU library. For NPS-PLA2, the PBDC aptamer 2692-74 BndUwas used for complex formation in SELEX with a TrpdU library. After ninerounds of SELEX, the pools were sequenced and individual clones wereprepared synthetically (AB-H 50mers) and tested for binding.

Table 28 provides a summary for the aptamers to the metalloproteinase-12(MMP-12) protein. The nucleic acid molecule of clone 4496-60 is as anaptamer forty (40) nucleotides in length comprising C-5 modifiedpyrimidines, specifically BndU, and is capable of binding to the MMP-12protein.

TABLE 28 MMP-12-4496-60 complex SELEX pool 12048 (NapdU mod.) (n = 384evaluable sequences) K_(d) (nM) K_(d) (nM) # Clone ID F.P. w/Comp.Sandwich 9 12048-21 1.53 NT Yes 8 12048-18 2.81 NT Yes 7 12048-3  3.71NT Yes 7 12048-54 3.66 NT Yes 4  12048-104 2 72 NT Yes 3  12048-234 0.94NT No 346 NT NT NT NT NT, not tested

Table 29 provides a summary for the aptamers to the phospholipase A2(NPS-PLA2) protein. The nucleic acid molecule of clone 2692-74, is as anaptamer forty (40) nucleotides in length comprising C-5 modifiedpyrimidines, specifically BndU, and is capable of binding to theNPS-PLA2 protein.

TABLE 29 NPS-PLA2-2692-74 complex SELEX pool 12055 (TrpdU mod.) (n = 384evaluable sequences) K_(d) (nM) K_(d) (nM) # Clone ID F.P. w/Comp.Sandwich 12 12055-22 0.38 NT Yes 372 NT NT NT NT NT, not tested

Example 5: Equilibrium Binding Constants of Aptamer Pairs

This example provides the representative method for measuring theequilibrium binding constants (K_(d) values) for the DNA aptamer pairs.

In brief, each of the clones for a given target was separatelyimmobilized on a specific LumAvidin bead type. The beads were thenpooled and used for capture of the target, and each of the sandwichcandidate clones was used separately for detection. With respect to thenumber of aptamer sandwich candidates (ranging from 7-16 per target) forthe eight panel proteins (ANGPT2, TSP2, CRDL1, MATN2, GPVI, ESAM, C7 andPLG), this approach reduced the two-dimensional matrix of pair-wisescreening for functional aptamer sandwiches to one dimension, from 1116assays (15²+14²+16²+14²+9²+7²+8²+7²) to 90 assays (15+14+16+14+9+7+8+7).The multiplexed Luminex sandwich screening assay allowed the rapididentification of functional aptamer pairs, e.g., by plotting the netsignals as heat maps. For CRDL1 shown as an example in FIG. 2C, 16aptamers were tested in a pair-wise matrix as capture and detectionreagents, including 11 sequences containing TrpdU, 4 with 2NapdU, andthe aptamer with BndU. The menu aptamer 3362-61 for CRDL1 performed wellwhen used as a capture or as a detection agent in conjunction with anyof the new clones. In contrast, clones 7575-6 and 7575-19 served betteras detection agents (FIG. 2D). For comparison, sandwich assays using oneof the new clones (7575-2) with any other new clone produced lowersignals, and no pair was as good as the pair with the menu SOMAmer3362-61 (FIG. 2E). No signals were obtained with the same SOMAmer usedfor capture and detection.

Sandwich binding curves on the Luminex platform were generated for sevenof the eight proteins: ANGPT2 (Angiopoietin-2), TSP2, CRDL1, MATN2, C7,PLG (Plasminogen), and GPVI, as well as, MMP-12 and NPS-PLA2 (FIG. 2F).The apparent equilibrium binding constants (K_(d) values) of the newaptamers for the protein complexes with the cognate aptamer ranged from0.02-2.7 nM (see Table 30). Of note, for the MATN2 and C7 sandwichassay, the “sandwich” K_(d) values improved when the C-5 modifiedpyrimidine for the aptamer 1 (capture) and aptamer 2 (detection) weredifferent. Specifically, for MATN2, the sandwich K_(d) value improved byabout 2.5 fold (comparing a BndU capture aptamer and BndU detectionaptamer (2.19 nM K_(d)) with a BndU capture aptamer and TrpdU detectionaptamer (0.88 nM K_(d)); see Table 30), and for C7, the sandwich K_(d)value improved by about 6.3 fold (BndU capture aptamer and BndUdetection aptamer (8.56 nM K_(d)) with a BndU capture aptamer and 2NapdUdetection aptamer (1.35 nM K_(d)); see Table 30).

The Swiss Prot numbers for the proteins in Table 30 are as follows: ESAM(SwissProt # Q96AP7) and CdtA—binary toxin (SwissProt # Q9KH42), ANGPT2(SwissProt #015123), TSP2 (SwissProt # P35442), CRDL1 (SwissProt #Q9BU40), MATN2 (SwissProt #000339), C7 (SwissProt # P10643), PLG(SwissProt # P00747), GPVI (SwissProt # Q9HCN6), MMP-12 (SwissProt#39900), NPS-PLA2 (SwissProt #14555).

TABLE 30 Target Protein Aptamer 1 (capture) Aptamer 2 (detection)Sandwich Name Clone ID K_(d) (nM) Modified Clone ID K_(d) (nM)^(a)Modified K_(d) (nM)^(b) ANGPT2¹ 2602-2  0.07 BndU 7560-4  0.29 (0.59)TrpdU 1.90 TSP2¹ 3339-33 0.07 BndU 7574-53 0.08 (0.07) 2NapdU 0.02CRDL1¹ 3362-61 1.05 BndU 7575-2  0.38 (0.19) 2NapdU 0.20 MATN2¹ 3325-2 0.14 BndU 7571-31 5.31 (3.65) TrpdU 0.88 3532-8^(c ) 1.76 (0.94) BndU2.19 GPVI¹ 3194-36 0.04 BndU 7564-5  0.04 (0.02) TrpdU 0.12 ESAM¹2981-9  0.25 BndU 7581-41 0.03 (0.06) 2NapdU >10 C7¹ 2888-49 2.71 BndU7579-67 11.3 (0.13) 2NapdU 1.35  2888-68^(c) 2.08 (2.39) BndU 8.56 PLG¹4151-6  4.86 BndU 7567-64 13.1 (1.94) TrpdU 2.70 4151-5^(c ) 2.00 (1.89)BndU 2.20 CdtA² 4758-6  0.86 TrpdU 5579-12 0.97 (0.28) 2NapdU 0.52MMP-12¹ 4496-60 0.22 BndU 12048-54  3.66 (NT)  NapdU 1.10 NPS-PLA2¹2692-74 0.02 BndU 12055-22  0.38 (NT)  TrpdU 4.80 ¹Human protein; ² C.difficile protein ^(a)Determined in radiolabel assay. Values inparentheses are for the complex of the target with aptamer 1^(b)Determined in Luminex bead-based sandwich assay ^(c)Aptamer fromarchived sequences (without sandwich SELEX)

Besides the LumAvidin bead-based assay, we also evaluated some of theaptamer pairs in plate-based sandwich assays. With biotinylated aptamersas capture agents immobilized on streptavidin-coated plates, and asdetection agents in conjunction with streptavidin-HRP conjugate, targettitrations indicated differences in assay performance compared to thebead-based test. While the apparent K_(d) value for the C7 aptamer pairwas essentially identical in the two assay types, the TSP2 pairperformed better in the Luminex assay, and the plasminogen pair somewhatbetter in the plate assay (see FIG. 3).

Tables 31 and 32 provide a summary of the physical and functionalcharacteristics of the aptamers summarized in Table 30 that were used tomake ternary complexes (i.e., “aptamer sandwiches”), and for the captureand detection of a target.

A description of the eleven (11) aptamers used as a “capture aptamer”(or first aptamer) is provided in Table 31.

TABLE 31 # of C-5 Mods. Base Composition of 40- Length C-5 (% of 40-mermer Central Region (%) Target K_(d) Clone ID (nts.) Mod. C.R.) A C GProtein (nM) 2602-2  51 BndU 10 (25%) 37.5% 15.0% 22.5% ANGPT2¹ 0.073339-33 51 BndU 9 (22.5%) 20.0% 30.0% 27.5% TSP2¹ 0.07 3362-61 51 BndU16 (40%) 17.5% 20.0% 22.5% CRDL1¹ 1.05 3325-2  51 BndU 14 (35%) 27.5%22.5% 15.0% MATN2¹ 0.14 3194-36 51 BndU 11 (27.5%) 20.0% 17.5% 35.0%GPVI¹ 0.04 2981-9  51 BndU 15 (37.5%) 17.5% 15.0% 30.0% ESAM¹ 0.252888-49 58 BndU 16 (40%) 17.5% 20.0% 22.5% C7¹ 2.71 4151-6  51 BndU 11(27.5%) 22.5% 25.0% 25.0% PLG¹ 4.86 4758-6  48 TrpdU 10 (25%) 25.0%20.0% 30.0% CdtA² 0.86 4496-60 50 BndU 9 (22.5%) 25.0% 25.0% 27.5%MMP-12¹ 0.22 2692-74 50 BndU 17 (42.5%) 25.0% 15.0% 17.5% NPS-PLA2¹ 0.86“nts.” is nucleotides “Mod.” is modification “C.R.” is central region ofaptamer ¹Human protein; ² C. difficile protein

Generally, the aptamers that functioned as a “capture aptamer” (or firstaptamer) were from about 48 to about 58 nucleotides in length (or fromabout 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, or 58 nucleotides inlength). Each aptamer comprised a 40-mer (40 nucleotides in length)central region (the remaining nucleotides of the aptamer flanked the40-mer central region). The 40-mer central region comprises from about 9to about 16 (or from 9, 10, 11, 12, 13, 14, 15 or 16) BndU or TrpdU C-5modified pyrimidines. Alternatively, the 40-mer central region comprisesfrom about 22% to about 40% (or from 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39 or 40%) BndU or TrpdU C-5 modifiedpyrimidines. Further, the 40-mer central region of the “capture aptamer”comprises from about 37% to about 58% GC content (or from about 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57 or 58% GC content). The “capture aptamer” (or first aptamer)comprises a binding affinity for its target protein of from about 0.07nM to about 4.9 nM (or from about 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4,3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8 or4.9 nM).

A description of the fourteen (14) aptamers used as a “detectionaptamer” (or second aptamer) is provided in Table 30.

TABLE 32 # of C-5 Mods. Base Composition of 40- Length C-5 (% of 40-mermer Central Region (%) Target K_(d) Clone ID (nts.) Mod. C.R.) A C GProtein (nM) 7560-4 50 TrpdU 11 (27.5%) 27.5% 30.0% 15.0% ANGPT2¹ 0.29 7574-53 50 2NapdU 13 (32.5%) 25.0% 27.5% 15.0% TSP2¹ 0.08 7575-2 502NapdU 12 (30%) 32.5% 22.5% 15.0% CRDL1¹ 0.38  7571-31 50 TrpdU 15(37.5%) 22.5% 17.5% 22.5% MATN2¹ 5.31 3532-8 58 BndU 13 (32.5%) 20.0%20.0% 27.5% MATN2¹ 1.76 7564-5 50 TrpdU 10 (25%) 20.0% 30.0% 25.0% GPVI¹0.04  7581-41 50 2NapdU 14 (35%) 15.0% 25.0% 25.0% ESAM¹ 0.03  7579-6750 2NapdU 11 (27.5%) 20.0% 25.0% 27.5% C7¹ 11.3  2888-68 58 BndU 11(27.5%) 15.0% 20.0% 37.5% C7¹ 2.08  7567-64 50 TrpdU 15 (37.5%)  7.5%37.5% 17.5% PLG¹ 13.1 4151-5 58 BndU 14 (35%) 25.0% 25.0% 15.0% PLG¹2.00  5579-12 50 2NapdU 9 (22.5%) 35.0% 20.0% 22.5% CdtA² 0.97 12048-5450 NapdU 5 (12.5%) 32.5% 20.0% 35.0% MMP-12¹ 3.66 12055-22 50 TrpdU 11(27.5%)  25%  25% 22.5% NPS-PLA2¹ 0.38 “nts.” is nucleotides “Mod.” ismodification “C.R.” is central region of aptamer ¹Human protein; ² C.difficile protein

Generally, the aptamers that functioned as a “detection aptamer” (orsecond aptamer) were from about 50 to about 58 nucleotides in length (orfrom about 50, 51, 52, 53, 54, 55, 56, 57 or 58 nucleotides in length).Each aptamer comprised a 40-mer (40 nucleotides in length) centralregion (the remaining nucleotides of the aptamer flanked the 40-mercentral region). The 40-mer central region comprises from about 5 toabout 15 (or from 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15) BndU, TrpdU,NapdU or 2NapdU C-5 modified pyrimidines. Alternatively, the 40-mercentral region comprises from about 12% to about 38% (or from 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37 or 38%) BndU, TrpdU, NapdU or 2NapdU C-5 modifiedpyrimidines. Further, the 40-mer central region of the “detectionaptamer” (or second aptamer) comprises from about 37% to about 58% GCcontent (or from about 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57 or 58% GC content). The “detectionaptamer” (or second aptamer) comprises a binding affinity for its targetprotein of from about 0.03 nM to about 13.1 nM (or from about 0.3, 0.35,0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1,3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6,4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1,9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.2, 10.4, 10.6, 10.8, 11,11.2, 11.4, 11.6, 11.8, 12, 12.2, 12.4, 12.6, 12.8, 13, 13.2, 13.4,13.6, 13.8 or 14 nM).

In summary, functional aptamer pairs suitable for sandwich assays ofprotein analytes were identified. Mining existing aptamers from archivedSELEX pools, combined with an approach of applying a second SELEX withtarget-aptamer complexes support the notion that the use of differenttypes of modified nucleotides enable for the identification of aptamerpairs capable of binding a target. In aptamer sandwich assays,background due to non-specific binding is reduced as a consequence ofdifferential off-rates between specific and non-specific aptamers. Thisfeature, combined with the added specificity inherent in two-reagentsandwich-type measurements, provides the basis for the development ofassays with greater specificity and higher multiplexing abilities.Aptamers pairs hold promise toward the development of specific panels invarious areas of medical diagnostics for which a large installed base ofinstruments is already in place.

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What is claimed is:
 1. A method for detecting a target in a sample, themethod comprising: a) contacting the sample with a first aptamer to forma mixture, wherein the first aptamer is capable of binding to the targetto form a first complex; b) incubating the mixture under conditions thatallow for the first complex to form; c) contacting the mixture with asecond aptamer, wherein the second aptamer is capable of binding thefirst complex to form a second complex; d) incubating the mixture underconditions that allow for the second complex to form; e) detecting forthe presence or absence of the first aptamer, the second aptamer, thetarget, the first complex or the second complex in the mixture, whereinthe presence of the first aptamer, the second aptamer, the target, thefirst complex or the second complex indicates that the target is presentin the sample; and wherein, the first aptamer comprises a first C-5pyrimidine modification scheme, the second aptamer comprises a secondC-5 pyrimidine modification scheme, and wherein the first C-5 pyrimidinemodification scheme and the second C-5 pyrimidine modification schemeare different, further wherein the first C-5 pyrimidine modificationscheme comprises a 5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU). 2.The method of claim 1, wherein the first aptamer has binding affinityfor the target and not the second aptamer.
 3. The method of claim 1,wherein the second aptamer has binding affinity for the target and notthe first aptamer.
 4. The method of claim 1, wherein the second aptamerhas binding affinity for the first complex.
 5. The method of claim 1,wherein the first aptamer binding region of the target and the secondaptamer binding region of the target are different regions.
 6. Themethod of claim 1, wherein the first aptamer and the second aptamer,independently, comprise RNA, DNA or a combination thereof.
 7. The methodof claim 1, wherein each uracil or thymine of the first aptamer is a5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU).
 8. The method of claim1, wherein the second C-5 pyrimidine modification scheme comprises a C-5modified pyrimidine selected from the group consisting of5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-[N-(1-naphthylmethyl)carboxyamide]-2′-deoxyuridine (NapdU),5-[N-(2-naphthylmethyl)carboxyamide]-2′-deoxyuridine (2-NapdU), and acombination thereof.
 9. The method of claim 1, wherein each uracil orthymine of the second aptamer is a C-5 modified pyrimidine selected fromthe group consisting of 5-(N-tryptaminocarboxyamide)-2′-deoxyuridine(TrpdU), 5-[N-(1-naphthylmethyl)carboxyamide]-2′-deoxyuridine (NapdU),5-[N-(2-naphthylmethyl)carboxyamide]-2′-deoxyuridine (2-NapdU), and acombination thereof.
 10. The method of claim 1, wherein the firstaptamer and the second aptamer, independently, are each from 20 to 100nucleotides in length.
 11. The method of claim 1, wherein the firstaptamer and/or the second aptamer further comprise a detectable moiety.12. The method of claim 11, wherein the detectable moiety is selectedfrom the group consisting of a dye, a quantum dot, a radiolabel, anelectrochemical functional group, an enzyme, an enzyme substrate, aligand and a receptor.
 13. The method of claim 1, wherein the targetcomprises a protein or a peptide.
 14. The method of claim 13, whereinthe target is a protein selected from the group consisting ANGPT2, TSP2,CRDL1, MATN2, GPVI, ESAM, C7, PLG, MMP-12, NPS-PLA2 and CdtA.
 15. Themethod of claim 1, wherein the dissociation constant (K_(d)) for thesecond complex is at least 0.02 nM, or from about 0.01 nM to about 10nM, or from about 0.02 nM to about 6 nM, or from about 0.02 nM to about3 nM.
 16. The method of claim 1, wherein the dissociation constant(K_(d)) for the first complex is from about 0.04 nM to about 5 nM, orfrom about 0.04 nM to about 4.8 nM.
 17. The method of claim 1, whereinthe dissociation constant (K_(d)) for the second aptamer and the targetis from about 0.03 nM to about 14 nM.
 18. A method comprising: a)contacting a target with a first aptamer to form a mixture, wherein thefirst aptamer is capable of binding the target to form a first complex;b) incubating the mixture under conditions that allow for the firstcomplex to form; c) contacting the mixture with a second aptamer,wherein the second aptamer is capable of binding the target to form asecond complex; d) incubating the mixture under conditions that allowfor the second complex to form; e) detecting for the presence or absenceof the first aptamer and the second aptamer in the mixture, wherein thepresence of both the first aptamer and second aptamer in the mixtureindicates that the binding of the first aptamer to the target and thebinding of the second aptamer to the target is non-competitive; andwherein, the first aptamer comprises a first C-5 pyrimidine modificationscheme, the second aptamer comprises a second C-5 pyrimidinemodification scheme, and wherein the first C-5 pyrimidine modificationscheme and the second C-5 pyrimidine modification scheme are different.19. The method of claim 18, wherein the first aptamer has bindingaffinity for the target and not the second aptamer.
 20. The method ofclaim 18, wherein the second aptamer has binding affinity for the targetand not the first aptamer.
 21. The method of claim 18, wherein thesecond aptamer has binding affinity for the first complex.
 22. Themethod of claim 18, wherein the first aptamer binding region of thetarget and the second aptamer binding region of the target are differentregions.
 23. The method of claim 18, wherein the first aptamer and thesecond aptamer, independently, comprise RNA, DNA or a combinationthereof.
 24. The method of claim 18, wherein each uracil or thymine ofthe first aptamer is a 5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU).25. The method of claim 18, wherein the second C-5 pyrimidinemodification scheme comprises a C-5 modified pyrimidine selected fromthe group consisting of 5-(N-tryptaminocarboxyamide)-2′-deoxyuridine(TrpdU), 5-[N-(1-naphthylmethyl)carboxyamide]-2′-deoxyuridine (NapdU),5-[N-(2-naphthylmethyl)carboxyamide]-2′-deoxyuridine (2-NapdU), and acombination thereof.
 26. The method of claim 18, wherein each uracil orthymine of the second aptamer is a C-5 modified pyrimidine selected fromthe group consisting of 5-(N-tryptaminocarboxyamide)-2′-deoxyuridine(TrpdU), 5-[N-(1-naphthylmethyl)carboxyamide]-2′-deoxyuridine (NapdU),5-[N-(2-naphthylmethyl)carboxyamide]-2′-deoxyuridine (2-NapdU), and acombination thereof.
 27. The method of claim 18, wherein the firstaptamer and the second aptamer, independently, are each from 20 to 100nucleotides in length.
 28. The method of claim 18, wherein the firstaptamer and/or the second aptamer further comprise a detectable moiety.29. The method of claim 28, wherein the detectable moiety is selectedfrom the group consisting of a dye, a quantum dot, a radiolabel, anelectrochemical functional group, an enzyme, an enzyme substrate, aligand and a receptor.
 30. The method of claim 18, wherein the targetcomprises a protein or a peptide.
 31. The method of claim 30, whereinthe target is a protein selected from the group consisting ANGPT2, TSP2,CRDL1, MATN2, GPVI, ESAM, C7, PLG, MMP-12, NPS-PLA2 and CdtA.
 32. Themethod of claim 18, wherein the dissociation constant (K_(d)) for thesecond complex is at least 0.02 nM, or from about 0.01 nM to about 10nM, or from about 0.02 nM to about 6 nM, or from about 0.02 nM to about3 nM.
 33. The method of claim 18, wherein the dissociation constant(K_(d)) for the first complex is from about 0.04 nM to about 5 nM, orfrom about 0.04 nM to about 4.8 nM.
 34. The method of claim 18, whereinthe dissociation constant (K_(d)) for the second aptamer and the targetis from about 0.03 nM to about 14 nM.
 35. The method of claim 18,wherein the first C-5 pyrimidine modification scheme comprises a C-5modified pyrimidine selected from the group consisting of5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),5-[N-(1-naphthylmethyl)carboxyamide]-2′-deoxyuridine (NapdU),5-[N-(2-naphthylmethyl)carboxyamide]-2′-deoxyuridine (2-NapdU), and acombination thereof.